Non-invasive ph-dependent imaging using quantitative chemical exchange saturation transfer (qcest)

ABSTRACT

In various embodiments, the invention teaches systems and methods for magnetic resonance imaging. In some embodiments, the invention teaches systems and methods for determining the source of pain in intervertebral discs by measuring one or more physiological biomarkers associated with disc pain and/or disc degeneration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 62/347,509 filed on Jun. 8, 2016,which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. AR066517awarded by National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to systems and methods forimaging and image processing.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Lower back pain is a major medical condition estimated to affect up to85% of the US population. Intervertebral disc (IVD) degeneration isoften associated with back pain. Although degenerate discs can beidentified using magnetic resonance imaging (MRI), they do not alwayscause pain. Therefore, if a patient with lower back pain has severaldegenerate discs, further examination is required to determine whichdisc is the source of the pain, prior to a decision of surgicalintervention. Standard procedures include discography, during which thesuspected discs are pressurized in order to provoke pain. This is apainful procedure that is also known to further accelerate discdegeneration, disc herniation, and loss of disc height and affect theadjacent endplates. It is also subjective to variations of the placementof the needle, pressure exerted, and anesthesia. Recent studies haveassociated low pH with discogenic pain. It is believed that pH couldpotentially serve as a new metabolic biomarker for discogenic back pain.

Chemical exchange saturation transfer (CEST) is an emerging MR (magneticresonance) technique to measure pH-dependent signal changes. Thistechnique exploits the constant chemical exchange, which ispH-sensitive, between water protons and solute protons in certainmolecules. The chemical exchange rate is dependent on pH values. Thesolute protons are first magnetization-saturated with a series offrequency selective radiofrequency (RF) pulses, and after exchangingwith water protons, the saturation is indirectly detected in the watersignal. For example, chemical exchange saturation transfer (CEST) is anemerging MR technique to detect glycosaminoglycan (GAG) content. Thistechnique exploits the constant chemical exchange between the waterprotons and the hydroxyl protons in GAG.

The hydroxyl protons will be first saturated, and after the transferwith water protons, the saturation will be indirectly detected in thewater signal. Previous studies have applied gagCEST to explore the GAGcontent distribution in patients with degenerative disc disease. Inaddition to concentration, correlation with pH was also reported.However, the gagCEST contrast is a rather complicated effect. Itinvolves multiple confounding factors, including but not limited to (a)exchange rate between water protons and GAG protons, which is dependenton the pH; (b) labile proton ratio, which is linearly correlated withGAG concentration; (c) water relaxation parameters T₁ and T₂; and (d)the RF irradiation power of CEST saturation module.

Recent studies have focused on separating the exchange rate or thelabile proton ratio from other confounding factors in the CESTexperiments. Among these methods, quantitative CEST (qCEST) allows forsimultaneous measurements of the exchange rate and labile proton ratio.It was developed based on the observation that the CEST effect can berepresented as a linear function of 1/B₁ ². Multiple CEST experimentswere performed with varying B₁ amplitudes for omega plot analysis.

Simultaneous measurements of pH value and concentration using qCEST havebeen shown in creatine phantom studies. Creatine protons have a slow tointermediate exchange rate with water protons. However, for GAG protonswhich undergo relatively faster chemical exchange, whether thistechnique can detect pH changes has not been investigated. In addition,most of the studies were performed on a preclinical scanner usingcontinuous-wave (cw) saturation pulse. No in vivo validation has beenperformed and potential clinical application is not yet clear.

There is clearly a need in the art for improved systems and methods fordiagnosing, prognosing, and monitoring the progression of conditionsinvolving tissue degeneration, and particularly those associated withback pain.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, compositions, articles ofmanufacture, and methods which are meant to be exemplary andillustrative, not limiting in scope.

In various embodiments, the present invention provides a method fordiagnosing a condition in a subject, the method comprising: imaging aregion of a subject's body with a magnetic resonance imaging (MRI)scanner, wherein the region is imaged using a quantitative chemicalexchange saturation transfer (qCEST) sequence; measuring one or morephysiological biomarkers within the imaged region, wherein thephysiological biomarkers comprise a labile proton exchange rate (k_(sw)between a solute pool and a water pool; and determining that the subjecthas the condition if the labile proton exchange rate is increasedrelative to a reference value. In some embodiments, the increased labileproton exchange rate is correlated to a low pH value. In someembodiments, the increased labile proton exchange rate is greater than200 exchanges/second. In some embodiments, the increased labile protonexchange rate is from 201 to 1000 exchanges/second. In some embodiments,the low pH value is from 5.6 to 6.99. In some embodiments, the referencevalue is a reference labile proton exchange rate, wherein the referencelabile proton exchange rate is from 100 to 200 exchanges/second. In someembodiments, the reference labile proton exchange rate is correlated toa reference pH value. In some embodiments, the reference pH value isfrom 7.0 to 7.2. In some embodiments, the condition is selected fromintervertebral disc degeneration, discogenic pain, discogenic low backpain, chronic low back pain, low back pain, back pain, chronic backpain, progressive intervertebral disc degeneration, osteoarthritis,rheumatoid arthritis, an articular cartilage injury, temporomandibulardisc degeneration and combinations thereof. In some embodiments, theimaged region of the subject's body comprises a joint or anintervertebral disc. In some embodiments, the condition is a painfulcondition. In some embodiments, the increased labile proton exchangerate is correlated with an upregulation of one or more pain-relatedfactors in the subject. In some embodiments, the one or morepain-related factors are selected from bradykinin receptor B1 (BDKRB1),calcitonin gene-related peptide (CGRP), and catechol-0-methyltransferase(COMT). In some embodiments, the increased labile proton exchange rateis correlated with an upregulation of one or more inflammation-relatedfactors in the subject. In some embodiments, the inflammation-relatedfactor is interleukin-6 (1L-6). In some embodiments, the increasedlabile proton exchange rate is correlated with an upregulation of one ormore neurogenic factors in the subject. In some embodiments, theneurogenic factor is brain-derived neurotrophic factor (BDNF) or nervegrowth factor (NGF). In some embodiments, the quantitative chemicalexchange saturation transfer (qCEST) sequence is a two dimension (2D)quantitative chemical exchange saturation transfer (qCEST) sequence.

In some embodiments, the quantitative chemical exchange saturationtransfer (qCEST) sequence is a three dimension (3D) quantitativechemical exchange saturation transfer (qCEST) sequence. In someembodiments, the MRI scanner is a 3.0T MRI scanner. In some embodiments,the MRI scanner is a 1.5T MRI scanner. In some embodiments, the MRIscanner is a 7.0T MRI scanner. In some embodiments, the method furthercomprises determining that an origin of the subject's condition iswithin the imaged region of the subject's body where the physiologicalbiomarker was measured. In some embodiments, the low pH value isindicative of the subject having the condition. In some embodiments, themethod further comprises selecting one or more treatments for thesubject if the condition is determined. In some embodiments, the methodfurther comprises treating the subject with one or more treatments ifthe condition is determined.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1A-FIG. 1B depict in accordance with various embodiments of theinvention, Ω-plots analysis of (FIG. 1A) phantoms with the sameconcentration (60 mM) but varying pH values (5.8, 6.1, 6.4, 6.7 and7.0); and (FIG. 1B) phantoms with the same pH value (7.0) but varyingGAG concentration (20 mM, 40 mM, 60 mM, 80 mM and 100 mM).

FIG. 2A-FIG. 21 ) depict in accordance with various embodiments of theinvention, quantitative results of the phantom study. (FIG. 2A)Pixel-wise mapping of labile proton exchange rate. (FIG. 2B) Pixel-wisemapping of labile proton ratio. (FIG. 2C) The chemical exchange rate asa function of pH. (FIG. 2D) The labile proton ratio as a function of GAGconcentration.

FIG. 3A-FIG. 3C depict in accordance with various embodiments of theinvention, representative images of IVDs and corresponding exchange ratemaps in one mini-pig. (FIG.¶3A) T2-weighted image in the sagittal plane.(FIG. 3B) Axial anatomical images of corresponding IVDs. (FIG. 3C)Exchange rate maps of corresponding IVDs. The IVDs with lower pH tend tohave higher exchange rates.

FIG. 4A-FIG. 4B depicts in accordance with various embodiments of theinvention, (FIG. 4A) 0-plots analysis of representative IVDs withvarying pH values (5.0, 5.8 and 6.7) and (FIG. 4B) the chemical exchangerate as a function of pH in the animal studies.

FIG. 5 depicts in accordance with various embodiments of the invention,a system including an MRI machine and a computing device, which arecapable of executing the inventive methods.

FIG. 6 depicts in accordance with various embodiments of the invention,IVD degeneration timeline. Minipigs underwent annular injury in four IVDlevels to induce degeneration. Following degeneration, animals wererandomly divided into 3 groups and scanned at 2, 6 and 10 weeks. At eachtime point, one of the groups was sacrificed and the pH within theinjured IVDs was measured. The IVDs were harvested for gene analyses andhistology

FIG. 7A-FIG. 7E depict in accordance with various embodiments of theinvention, IVD degeneration following intra-discal puncture. (FIG. 7A)The progress of IVD degeneration at 2, 6 and 10 weeks followingpuncture, as monitored by T₂-weighted sagittal MRI. White arrows denotethe degenerated IVDs. Quantification of (FIG. 7B) T2, (FIG. 7C) T_(i),and (FIG. 7D) T_(1p) mappings of degenerated IVDs compared to healthycontrols at 2, 6 and 10 weeks following puncture (n=12 per experimentalgroup; *p<0.05, ****p<0.0001). (FIG. 7E) Hematoxylin and eosin stainingof representative IVDs that underwent degeneration at 2, 6 and 10 weeksafter induction of degeneration, at low magnification (upper subfigures;scale bars, 1 mm) and high magnification (lower subfigures; scale bars,100 μm).

FIG. 8A-FIG. 8D depict in accordance with various embodiments of theinvention, pH and qCEST changes following 1VD degeneration. (FIG. 8A)Correlation between the qCEST signal represented by the exchange ratebetween solute pool and water pool (k_(SW)) and the pH measured withinthe IVD following animal sacrifice. (FIG. 8B) ROC curve analysis ofqCEST signaling for the detection of degenerating 1VDs. (FIG. 8C) pH and(FIG. 8D) qCEST measurements within the degenerating IVDs at 2, 6 and 10weeks after degeneration. (n=12 per experimental group; *p<0.05,**p<0.01, ****p=0.0001; qCEST=quantitative chemical exchange saturationtransfer).

FIG. 9A-FIG. 9E depict in accordance with various embodiments of theinvention, Pain and inflammatory markers upregulation in degeneratingIVDs. Quantitative RT-PCR analysis of (FIG. 9A-FIG. 9C) pain-relatedgenes (CGRP, BDKRB1 and COMT), (FIG. 9D) IL-6 and (FIG. 9E) BDNFharvested from the annulus fibrosus and nucleus pulposus of degeneratedIVDs at 2, 6 and 10 weeks after induction of degeneration. (n=3 pergroup; *p<0.05, **p<0.01; CGRP=calcitonin gene-related peptide,BDKRB1=Bradykinin receptor B1, COMT=catechol-0-methyltransferase,BDNF=brain-derived neurotrophic factor).

FIG. 10 depicts in accordance with various embodiments of the invention,Immunofluorescence analysis of IVD degeneration and marker upregulation.Immunostaining of serial slides of nucleus pulposus from weeks 2, 6 and10 after degeneration against COMT, IL-6, BDNF, CGRP, BDKRB1 andcounterstaining with DAPI. Merged panels of the different stainings arepresented on the right column. (NP=nucleus pulposus, CGRP=calcitoningene-related peptide, BDKRB1=Bradykinin receptor B1,COMT=catechol-0-methyltransferase, BDNF=brain-derived neurotrophicfactor)

FIG. 11A-FIG. 11E depict in accordance with various embodiments of theinvention, Linear correlation between qCEST and biomarkers indegenerating IVDs. Correlation curves between qCEST signal andcorresponding expression of (FIG. 11A) CGRP, (FIG. 11B) BDKRB1, (FIG.11C) COMT, (FIG. 11D) IL-6 and (FIG. 11E) BDNF extracted fromdegenerated and healthy IVDs.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Westbrook et al., MRI in Practice 4^(th) ed., andGuyton and Hall, Textbook of Medical Physiology 12 ^(th) ed., provideone skilled in the art with a general guide to many of the terms used inthe present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Other features and advantages of theinvention will become apparent from the following detailed description,taken in conjunction with the accompanying drawings, which illustrate,by way of example, various features of embodiments of the invention.Indeed, the present invention is in no way limited to the methods andmaterials described. For convenience, certain terms employed herein, inthe specification, examples and appended claims are collected here.

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, systems, articles of manufacture, andrespective component(s) thereof, that are useful to an embodiment, yetopen to the inclusion of unspecified elements, whether useful or not. Itwill be understood by those within the art that, in general, terms usedherein are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.).

Unless stated otherwise, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of claims) can be construedto cover both the singular and the plural. The recitation of ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate value falling within the range.Unless otherwise indicated herein, each individual value is incorporatedinto the specification as if it were individually recited herein. Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (for example,“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the application and does not pose alimitation on the scope of the application otherwise claimed. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.” No language in thespecification should be construed as indicating any non-claimed elementessential to the practice of the application.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” when used in reference to a disease, disorder, condition,disease condition, or medical condition, refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to reverse, alleviate, ameliorate, inhibit, lessen, slow down or stopthe progression or severity of a symptom or condition. The term“treating” includes reducing or alleviating at least one adverse effector symptom of a condition. Treatment is generally “effective” if one ormore symptoms or clinical markers are reduced. Alternatively, treatmentis “effective” if the progression of a disease, disorder, condition,disease condition, or medical condition is reduced or halted. That is,“treatment” includes not just the improvement of symptoms or markers,but also a cessation or at least slowing of progress or worsening ofsymptoms that would be expected in the absence of treatment. Also,“treatment” may mean to pursue or obtain beneficial results, or lowerthe chances of the individual developing the condition even if thetreatment is ultimately unsuccessful. Those in need of treatment includethose already with the condition as well as those prone to have thecondition or those in whom the condition is to be prevented.Non-limiting examples of treatments or therapeutic treatments includepharmacological, biological, cell and gene therapies and/orinterventional surgical treatments. Non-limiting examples of a treatmentor therapeutic treatment are pharmacological treatments. Non-limitingexamples of a treatment or therapeutic treatment are biologicaltreatments. Non-limiting examples of a treatment or therapeutictreatment are cell treatments. Non-limiting examples of a treatment ortherapeutic treatment are gene therapies. Non-limiting examples of atreatment or therapeutic treatment are interventional surgicaltreatments. A treatment or therapeutic treatment may include one or moretreatments or a combination of treatments.

“Beneficial results” or “desired results” may include, but are in no waylimited to, lessening or alleviating the severity of the disease,disorder, condition, disease condition, or medical condition, preventingthe disease, disorder, condition, disease condition, or medicalcondition from worsening, curing the disease, disorder, condition,disease condition, or medical condition, preventing the disease,disorder, condition, disease condition, or medical condition fromdeveloping, lowering the chances of a patient developing the disease,disorder, condition, disease condition, or medical condition, decreasingmorbidity and mortality, and prolonging a patient's life or lifeexpectancy. As non-limiting examples, “beneficial results” or “desiredresults” may be alleviation of one or more symptom(s), diminishment ofextent of the deficit, stabilized (i.e., not worsening) state of adisease, disorder, condition, disease condition, or medical condition,delay or slowing of a disease, disorder, condition, disease condition,or medical condition, and amelioration or palliation of symptomsassociated with a disease, disorder, condition, disease condition, ormedical condition.

As used herein, the term “administering,” refers to the placement anagent or a treatment as disclosed herein into a subject by a method orroute which results in at least partial localization of the agent ortreatment at a desired site. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, via inhalation, oral, anal, intra-anal, peri-anal,transmucosal, transdermal, parenteral, enteral, topical or local.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intracranial, intraventricular,intrathecal, epidural, intradural, intraorbital, infusion,intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravascular, intravenous, intraarterial, subarachnoid,subcapsular, subcutaneous, transmucosal, or transtracheal.

“Diagnostic” means identifying the presence or nature of a pathologiccondition and includes identifying patients who are at risk ofdeveloping a specific disease, disorder, condition, disease condition,or medical condition. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of “true positives”).Diseased individuals not detected by the assay are “false negatives.”Subjects who are not diseased and who test negative in the assay, aretermed “true negatives.” The “specificity” of a diagnostic assay is 1minus the false positive rate, where the “false positive” rate isdefined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

By “at risk of” is intended to mean at increased risk of, compared to anormal subject, or compared to a control group, e.g. a patientpopulation. Thus a subject carrying a particular marker may have anincreased risk for a specific disease, disorder, condition, diseasecondition, or medical condition, and be identified as needing furthertesting. “Increased risk” or “elevated risk” mean any statisticallysignificant increase in the probability, e.g., that the subject has thedisease, disorder, condition, disease condition, or medical condition.The risk is preferably increased by at least 10%, more preferably atleast 20%, and even more preferably at least 50% over the control groupwith which the comparison is being made.

The term “statistically significant” or “significantly” refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true. The decision is often made using thep-value.

The terms “detection”, “detecting” and the like, may be used in thecontext of detecting a disease, disorder, condition, disease condition,or medical condition (e.g. when positive assay results are obtained). Inthe latter context, “detecting” and “diagnosing” are consideredsynonymous.

The term “diagnosis,” or “dx,” refers to the identification of thenature and cause of a certain phenomenon. As used herein, a diagnosistypically refers to a medical diagnosis, which is the process ofdetermining which disease, disorder, condition, disease condition, ormedical condition explains a symptoms and signs. A diagnostic procedure,often a diagnostic test or assay, can be used to provide a diagnosis. Adiagnosis can comprise detecting the presence of a disease, disorder,condition, disease condition, or medical condition or the risk ofgetting a disease, disorder, condition, disease condition, or medicalcondition.

The term “prognosis,” or “px,” as used herein refers to predicting thelikely outcome of a current standing. For example, a prognosis caninclude the expected duration and course of a disease, disorder,condition, disease condition, or medical condition, such as progressivedecline or expected recovery.

The term “theranosis,” or “ix” as used herein refers to a diagnosis orprognosis used in the context of a medical treatment. For example,theranostics can include diagnostic testing used for selectingappropriate and optimal therapies (or the inverse) based on the contextof genetic content or other molecular or cellular analysis. Theranosticsincludes pharmacogenomics, personalized and precision medicine.

As used herein, a “subject” means a human or animal. For example, theanimal is a vertebrate such as a primate, rodent, domestic animal orgame animal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus.

Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.Domestic and game animals include cows, horses, pigs, deer, bison,buffalo, feline species, e.g., domestic cat, and canine species, e.g.,dog, fox, wolf. The terms, “patient”, “individual” and “subject” areused interchangeably herein. In an embodiment, the subject is mammal.The mammal can be a human, non-human primate, mouse, rat, dog, cat,horse, or cow, but are not limited to these examples. In addition, themethods described herein can be used to treat domesticated animalsand/or pets. In an embodiment, the subject is a human.

“Mammal,” as used herein, refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domesticated mammals, such asdogs and cats; laboratory animals including rodents such as mice, ratsand guinea pigs, and the like. The term does not denote a particular ageor sex. Thus, adult and newborn subjects, whether male or female, areintended to be included within the scope of this term. Unless otherwiseindicated, the subjects described herein can include mammals.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a disease, disorder, condition,disease condition, or medical condition in need of treatment or one ormore complications related to the disease, disorder, condition, diseasecondition, or medical condition, and optionally, have already undergonetreatment for the disease, disorder, condition, disease condition, ormedical condition or the one or more complications related to thedisease, disorder, condition, disease condition, or medical condition.Alternatively, a subject can also be one who has not been previouslydiagnosed as having a disease, disorder, condition, disease condition,or medical condition or one or more complications related to thedisease, disorder, condition, disease condition, or medical condition.For example, a subject can be one who exhibits one or more risk factorsfor a disease, disorder, condition, disease condition, or medicalcondition or one or more complications related to the disease, disorder,condition, disease condition, or medical condition or a subject who doesnot exhibit risk factors. For example, a subject can be one who exhibitsone or more symptoms for a disease, disorder, condition, diseasecondition, or medical condition or one or more complications related tothe disease, disorder, condition, disease condition, or medicalcondition or a subject who does not exhibit symptoms. A “subject inneed” of diagnosis or treatment for a particular disease, disorder,condition, disease condition, or medical condition can be a subjectsuspected of having that disease, disorder, condition, diseasecondition, or medical condition, diagnosed as having that disease,disorder, condition, disease condition, or medical condition, alreadytreated or being treated for that disease, disorder, condition, diseasecondition, or medical condition, not treated for that disease, disorder,condition, disease condition, or medical condition, or at risk ofdeveloping that disease, disorder, condition, disease condition, ormedical condition.

“Sample” is used herein in its broadest sense. The term “biologicalsample” as used herein denotes a sample taken or isolated from abiological organism. A sample or biological sample may comprise a bodilyfluid including blood, serum, plasma, tears, aqueous and vitreous humor,spinal fluid; a soluble fraction of a cell or tissue preparation, ormedia in which cells were grown; or membrane isolated or extracted froma cell or tissue; polypeptides, or peptides in solution or bound to asubstrate; a cell; a tissue; a tissue print; a fingerprint, skin orhair; fragments and derivatives thereof. Non-limiting examples ofsamples or biological samples include cheek swab; mucus; whole blood,blood, serum; plasma; urine; saliva; semen; lymph; fecal extract;sputum; other body fluid or biofluid; cell sample; and tissue sampleetc. The term also includes a mixture of the above-mentioned samples orbiological samples. The term “sample” also includes untreated orpretreated (or pre processed) biological samples. In some embodiments, asample or biological sample can comprise one or more cells from thesubject. In some embodiments, a sample or biological sample can compriseone or more tissue samples from the subject.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Upregulation” means an increase in gene expression.

“Downregulation” means a decrease in gene expression.

Methods of the Invention

In various embodiments, the invention provides a method comprisingimaging a region of a subject's body with a magnetic resonance imaging(MRI) scanner; and measuring one or more physiological biomarkers withinthe imaged region, wherein (a) the physiological biomarkers include alabile proton ratio (f_(r)) between a solute pool and a water pooland/or an exchange rate (k_(5w).) between the solute pool and the waterpool. In some embodiments, the region is imaged using a quantitativechemical exchange saturation transfer (qCEST) sequence.

In various embodiments, the invention provides method for diagnosing acondition in a subject, the method comprising: imaging a region of asubject's body with a magnetic resonance imaging (MRI) scanner, whereinthe region is imaged using a quantitative chemical exchange saturationtransfer (qCEST) sequence; and measuring one or more physiologicalbiomarkers within the imaged region, wherein the physiologicalbiomarkers comprise a labile proton exchange rate (k_(sw)) between asolute pool and a water pool

In various embodiments, the invention provides method for diagnosing acondition in a subject, the method comprising: imaging a region of asubject's body with a magnetic resonance imaging (MRI) scanner, whereinthe region is imaged using a quantitative chemical exchange saturationtransfer (qCEST) sequence; measuring one or more physiologicalbiomarkers within the imaged region, wherein the physiologicalbiomarkers comprise a labile proton exchange rate (k_(sw)) between asolute pool and a water pool; and determining that the subject has thecondition if the labile proton exchange rate is increased relative to areference value. In some embodiments, the method further comprisesselecting one or more treatments for the subject if the condition isdetermined. In some embodiments, the treatments are selected frompharmacological, biological, cell and gene therapies and/orinterventional surgical treatments. In some embodiments, the increasedlabile proton exchange rate is correlated to a low pH value. In someembodiments, the increased labile proton exchange rate is greater than200 exchanges/second. In some embodiments, the increased labile protonexchange rate is from 201 to 1000 exchanges/second. In some embodiments,the low pH value is from 5.6 to 6.99. In some embodiments, the referencevalue is a reference labile proton exchange rate, wherein the referencelabile proton exchange rate is from 100 to 200 exchanges/second. In someembodiments, the reference labile proton exchange rate is correlated toa reference pH value. In some embodiments, the reference pH value isfrom 7.0 to 7.2 In some embodiments, the condition is selected fromintervertebral disc degeneration, discogenic pain, discogenic low backpain, chronic low back pain, low back pain, back pain, chronic backpain, progressive intervertebral disc degeneration, osteoarthritis,rheumatoid arthritis, an articular cartilage injury, temporomandibulardisc degeneration and combinations thereof. In some embodiments, theimaged region of the subject's body comprises a joint or anintervertebral disc. In some embodiments, the condition is a painfulcondition. In some embodiments, the increased labile proton exchangerate is correlated with an upregulation of one or more pain-relatedfactors in the subject. In some embodiments, the one or morepain-related factors are selected from bradykinin receptor B1 (BDKRB1),calcitonin gene-related peptide (CGRP), and catechol-0-methyltransferase(COMT). In some embodiments, the increased labile proton exchange rateis correlated with an upregulation of one or more inflammation-relatedfactors in the subject. In some embodiments, the inflammation-relatedfactor is interleukin-6 (IL-6). In some embodiments, the increasedlabile proton exchange rate is correlated with an upregulation of one ormore neurogenic factors in the subject. In some embodiments, theneurogenic factor is brain-derived neurotrophic factor (BDNF) or nervegrowth factor (NGF). In some embodiments, the quantitative chemicalexchange saturation transfer (qCEST) sequence is a two dimension (2D)quantitative chemical exchange saturation transfer (qCEST) sequence. Insome embodiments, the quantitative chemical exchange saturation transfer(qCEST) sequence is a three dimension (3D) quantitative chemicalexchange saturation transfer (qCEST) sequence. In some embodiments, theMRI scanner is a 3.0T MRI scanner. In some embodiments, the MRI scanneris a 1.5T MRI scanner. In some embodiments, the MRI scanner is a 7.0TMRI scanner. In some embodiments, further comprising determining that anorigin of the subject's condition is within the imaged region of thesubject's body where the physiological biomarker was measured. In someembodiments, the low pH value is indicative of the subject having thecondition. In some embodiments, the method further comprises selectingone or more treatments for the subject if the condition is determined.In some embodiments, the method further comprises treating the subjectwith one or more treatments if the condition is determined.

In various embodiments, the invention provides a method for treating asubject diagnosed with a condition, wherein the subject was diagnosedwith the condition by a method comprising: imaging a region of asubject's body with a magnetic resonance imaging (MRI) scanner, whereinthe region is imaged using a quantitative chemical exchange saturationtransfer (qCEST) sequence; measuring one or more physiologicalbiomarkers within the imaged region, wherein the physiologicalbiomarkers comprise a labile proton exchange rate (k_(SW)) between asolute pool and a water pool; determining that the subject has thecondition if the labile proton exchange rate is increased relative to areference value; selecting a treatment for the subject; and treating thesubject with the treatment.

In various embodiments, the invention provides a method for treating asubject diagnosed with a condition, wherein the subject was diagnosedwith the condition by a method comprising: imaging a region of asubject's body with a magnetic resonance imaging (MRI) scanner, whereinthe region is imaged using a quantitative chemical exchange saturationtransfer (qCEST) sequence; measuring one or more physiologicalbiomarkers within the imaged region, wherein the physiologicalbiomarkers comprise a labile proton ratio (f_(r)) between a solute pooland a water pool and/or an exchange rate (k_(SW)) between the solutepool and the water pool; selecting a treatment for the subject; andtreating the subject with the treatment.

In various embodiments, the invention provides a method for treating asubject diagnosed with a condition, comprising requesting the diagnosisresults so that the subject may be treated; selecting a treatment forthe subject; and treating the subject based on the diagnosis results,wherein the subject was diagnosed with the condition by a methodcomprising: imaging a region of a subject's body with a magneticresonance imaging (MRI) scanner, wherein the region is imaged using aquantitative chemical exchange saturation transfer (qCEST) sequence;measuring one or more physiological biomarkers within the imaged region,wherein the physiological biomarkers comprise a labile proton exchangerate (k_(SW)) between a solute pool and a water pool; and determiningthat the subject has the condition if the labile proton exchange rate isincreased relative to a reference value.

In various embodiments, the invention provides a method for treating asubject diagnosed with a condition, comprising requesting the diagnosisresults so that the subject may be treated; selecting a treatment forthe subject; and treating the subject based on the diagnosis results,wherein the subject was diagnosed with the condition by a methodcomprising: imaging a region of a subject's body with a magneticresonance imaging (MRI) scanner, wherein the region is imaged using aquantitative chemical exchange saturation transfer (qCEST) sequence; andmeasuring one or more physiological biomarkers within the imaged region,wherein the physiological biomarkers comprise a labile proton ratio(f_(r)) between a solute pool and a water pool and/or an exchange rate(k_(SW)) between the solute pool and the water pool.

A method for prognosing a condition in a subject, the method comprising:imaging a region of a subject's body with a magnetic resonance imaging(MRI) scanner, wherein the region is imaged using a quantitativechemical exchange saturation transfer (qCEST) sequence; measuring one ormore physiological biomarkers within the imaged region, wherein thephysiological biomarkers comprise a labile proton exchange rate (k_(SW))between a solute pool and a water pool; and comparing a measurement ofone or more physiological biomarkers to a previous measurement of thesame one or more physiological biomarkers, wherein an increase in thelabile proton exchange rate over time is a poor prognosis of thecondition. In some embodiments, the method further comprises selectingone or more treatments for the subject based on the prognosis of thecondition. In some embodiments, the method further comprises treatingthe subject with one or more treatments based on the prognosis of thecondition. In some embodiments, a low pH value is indicative of a poorprognosis of the condition.

A method for prognosing a condition associated with tissue degenerationand/or pain in a subject, comprising: imaging a region of a subject'sbody, wherein the region is imaged using a quantitative chemicalexchange saturation transfer (qCEST) sequence; measuring one or morephysiological biomarkers within the imaged region, wherein thephysiological biomarkers comprise a labile proton exchange rate (k_(SW))between a solute pool and a water pool; and prognosing the condition bycomparing measurements of one or more physiological biomarkers measuredwithin the imaged region to previous measurements of the same one ormore physiological biomarkers measured within the imaged region.

A method for prognosing a condition associated with tissue degenerationand/or pain in a subject, comprising: imaging a region of a subject'sbody, wherein the region is imaged using a quantitative chemicalexchange saturation transfer (qCEST) sequence; measuring one or morephysiological biomarkers within the imaged region, wherein thephysiological biomarkers comprise a labile proton ratio (f_(r)) betweena solute pool and a water pool and/or an exchange rate (k_(SW)) betweenthe solute pool and the water pool; and prognosing the condition bycomparing measurements of one or more physiological biomarkers measuredwithin the imaged region to previous measurements of the same one ormore physiological biomarkers measured within the imaged region.

In various embodiments, the invention provides method for detecting acondition in a subject, the method comprising: imaging a region of asubject's body with a magnetic resonance imaging (MRI) scanner, whereinthe region is imaged using a quantitative chemical exchange saturationtransfer (qCEST) sequence; measuring one or more physiologicalbiomarkers within the imaged region, wherein the physiologicalbiomarkers comprise a labile proton exchange rate (k_(SW)) between asolute pool and a water pool; and determining that the subject has thecondition if the labile proton exchange rate is increased relative to areference value. In some embodiments, the method further comprisesselecting one or more treatments for the subject if the condition isdetermined. In some embodiments, the treatments are selected frompharmacological, biological, cell and gene therapies and/orinterventional surgical treatments. In some embodiments, the methodfurther comprises treating the subject with one or more treatments ifthe condition is determined.

In various embodiments, the invention provides method for detecting acondition in a subject, the method comprising: imaging a region of asubject's body with a magnetic resonance imaging (MRI) scanner, whereinthe region is imaged using a quantitative chemical exchange saturationtransfer (qCEST) sequence; measuring one or more physiologicalbiomarkers within the imaged region, wherein the physiologicalbiomarkers comprise a labile proton ratio (f_(r)) between a solute pooland a water pool and/or an exchange rate (k_(SW)) between the solutepool and the water pool. In some embodiments, the method furthercomprises selecting one or more treatments for the subject if thecondition is determined. In some embodiments, the treatments areselected from pharmacological, biological, cell and gene therapiesand/or interventional surgical treatments. In some embodiments, themethod further comprises treating the subject with one or moretreatments if the condition is determined.

In some embodiments, the one or more magnetic resonance images areobtained over a period of time. In some embodiments, the one or moremagnetic resonance images are obtained at different times. In someembodiments, the one or more magnetic resonance images are obtainedcontemporaneously. In some embodiments, the one or more magneticresonance images are two or more magnetic resonance images. In someembodiments, the period of time is measured in milliseconds, seconds,minutes, hours, days, months, or years, or combinations thereof.

In some embodiments, the invention provides a method for determining therisk of developing a condition in a subject, comprising imaging a regionof a subject's body with a magnetic resonance imaging (MRI) scanner,wherein the region is imaged using a quantitative chemical exchangesaturation transfer (qCEST) sequence; measuring one or morephysiological biomarkers within the imaged region, wherein thephysiological biomarkers include a labile proton exchange rate (k_(SW))between a solute pool and a water pool; and comparing the labile protonexchange rate from the subject to a reference value, wherein an increasein the labile exchange rate from the subject compared to the referencevalue is indicative of an increased risk of the subject developing thecondition. In some embodiments, the low pH value is indicative of theincreased risk of the subject developing the condition. In someembodiments, the method further comprises selecting one or moretreatments for the subject based on the increased risk of the subjectdeveloping the condition. In some embodiments, the method furthercomprises treating the subject with one or more treatments based on theincreased risk of the subject developing the condition.

In some embodiments, the method further comprises comparing the labileproton exchange rate from the subject to a reference value, wherein anincrease in the labile exchange rate from the subject compared to thereference value is an assessment of the subject, wherein the assessmentis a prognosis of developing a condition. In some embodiments, themethod further comprises comparing the labile proton exchange rate fromthe subject to a reference value, wherein an increase in the labileexchange rate from the subject compared to the reference value is anassessment of the subject, wherein the assessment is a diagnosis of acondition. In some embodiments, the method further comprises comparingthe labile proton exchange rate from the subject to a reference value,wherein an increase in the labile exchange rate from the subjectcompared to the reference value is an assessment of the subject isindicative of a condition. In some embodiments, the method furthercomprises treating the subject based on the assessment. In someembodiments, the method further comprises selecting one or moretreatments for the subject based on the assessment. In some embodiments,the method further comprises treating the subject with one or moretreatments based on the assessment.

In some embodiments, the invention provides a method for determining therisk of developing a condition in a subject, comprising imaging a regionof a subject's body with a magnetic resonance imaging (MRI) scanner,wherein the region is imaged using a quantitative chemical exchangesaturation transfer (qCEST) sequence; measuring one or morephysiological biomarkers within the imaged region, wherein thephysiological biomarkers include a labile proton exchange rate (k_(SW))between a solute pool and a water pool; and comparing the labile protonexchange rate from the subject to a reference value, wherein an increasein the labile exchange rate from the subject compared to the referencevalue is indicative of an increased risk of the subject developing thecondition. In some embodiments, the low pH value is indicative of theincreased risk of the subject developing the condition. In someembodiments, the method further comprises selecting one or moretreatments for the subject based on the increased risk of the subjectdeveloping the condition. In some embodiments, the method furthercomprises treating the subject with one or more treatments based on theincreased risk of the subject developing the condition

In some embodiments, the increased labile proton exchange rate iscorrelated with an upregulation of one or more pain-related factors inthe subject. In some embodiments, the pain-related factors (pain-relatedmarkers) are selected from bradykinin receptor B1 (BDKRB1), calcitoningene-related peptide (CGRP) and catechol-0-methyltransferase (COMT). Insome embodiments, the pain-related factor is bradykinin receptor B1(BDKRB1). In some embodiments, the pain-related factor is calcitoningene-related peptide (CGRP). In some embodiments, the pain-relatedfactor is catechol-0-methyltransferase (COMT). In some embodiments, themethod further comprises determining that an origin of the subject'spain associated with the condition is within the region of the subject'sbody where the expression of one or more pain-related factors(pain-related markers) is detected. In some embodiments, the conditionis a painful condition.

In some embodiments, the increased labile proton exchange rate iscorrelated with an upregulation of one or more inflammation-relatedfactors in the subject. In some embodiments, the inflammatory factors(inflammation-related markers or inflammation-related factors) areselected from interleukin-6 (IL-6). In some embodiments, the methodfurther comprises determining that an origin of the subject's painassociated with the condition is within the region of the subject's bodywhere the expression of one or more inflammatory factors(inflammation-related markers) is detected. In some embodiments, thecondition is a painful condition.

In some embodiments, the increased labile proton exchange rate iscorrelated with an upregulation of one or more neurogenic factors in thesubject. In some embodiments, the neurogenic factors (neurogenicmarkers) are selected from brain-derived neurotrophic factor (BDNF) andnerve growth factor (NGF). In some embodiments, the method furthercomprises determining that an origin of the subject's pain associatedwith the condition is within the region of the subject's body where theexpression of one or more neurogenic factors (neurogenic markers) isdetected. In some embodiments, the condition is a painful condition

In some embodiments, the physiological biomarkers comprise a labileproton ratio (f_(r)) between a solute pool and a water pool and/or anexchange rate (k_(SW)) between the solute pool and the water pool. Insome embodiments, the physiological biomarkers comprise a labile protonratio (f_(r)) between a solute pool and a water pool and/or a labileproton exchange rate (k_(SW)) between the solute pool and the waterpool. In some embodiments, the physiological biomarkers comprise alabile proton ratio (f_(r)) between a solute pool and a water pool and alabile proton exchange rate (k_(SW)) between the solute pool and thewater pool. In some embodiments, the physiological biomarkers comprise alabile proton ratio (f_(r)) between a solute pool and a water pool or alabile proton exchange rate (k_(s,w)) between the solute pool and thewater pool. In some embodiments, the physiological biomarkers comprise alabile proton ratio (f_(r)) between a solute pool and a water pool. Insome embodiments, the physiological biomarkers comprise an exchange rate(k_(SW)) between a solute pool and a water pool. In some embodiments,the physiological biomarkers comprise a labile proton exchange rate(k_(sw)) between a solute pool and a water pool.

In certain embodiments, the abnormal physiological states may include,but are in no way limited to, low pH and/or low GAG concentrationcompared to a normal subject without the condition. In certainembodiments, the abnormal physiological state is low GAG concentrationcompared to a normal subject without the condition. In certainembodiments, the abnormal physiological state is low GAG concentration.In some embodiments, the abnormal physiological state is low pH comparedto a normal subject without the condition. In some embodiments, the GAGconcentration is relative to a healthy, non-degenerate disc. In someembodiments, the low pH concentration is relative to a healthy,non-degenerate disc. In some embodiments, the GAG concentration isrelative to a healthy, non-degenerate disc in the subject. In someembodiments, the low pH concentration is relative to a healthy,non-degenerate disc in the subject. In some embodiments, the normalsubject is the subject, wherein the subject does not have a condition.In some embodiments, the normal subject is the subject before thesubject is treated for a condition. In some embodiments, the normalsubject is the subject that has been treated for a condition. In someembodiments, the normal subject is the subject at an earlier time point(earlier point in time).

In certain embodiments, the abnormal physiological state is low pHcompared to a normal subject without the condition. In certainembodiments, the abnormal physiological state is low pH. In someembodiments, the abnormal physiological state is low pH compared to a pHvalue obtained from a pH reference sample (reference pH value). In someembodiments, low pH (low pH value) is 5.6-6.99. In some embodiments, lowpH (low pH value) is5.6-5.7,5.6-5.8,5.6-5.9,5.6-6.0,5.6-6.1,5.6-6.2,5.6-6.3,5.6-6.4,5.6-6.5,5.6-6.6,5.6-6.7,5.6-6.8,5.6-6.9, or 5.6-6.99.

In some embodiments, low pH (low pH value) is 5.60 to 6.99, 5.60 to6.90, 5.60 to 6.80, 5.60 to 6.70, 5.60 to 6.60, 5.60 to 6.50, 5.60 to6.40, 5.60 to 6.30, 5.60 to 6.20, 5.60 to 6.10, 5,60 to 6.00, 5.60 to5.90, 5.60 to 5.80, 5.60 to 5.70, 5.70 to 6.99, 5.70 to 6.90, 5.70 to6.80, 5.70 to 6.70, 5.70 to 6.60, 5.70 to 6.50, 5.70 to 6.40, 5.70 to6.30, 5.70 to 6.20, 5.70 to 6.10, 5.70 to 6.00, 5.70 to 5.90, 5.70 to5.80, 5.80 to 6.99, 5.80 to 6.90, 5.80 to 6.80, 5.80 to 6.70, 5.80 to6.60, 5.80 to 6.50, 5.80 to 6.40, 5.80 to 6.30, 5.80 to 6.20, 5.80 to6.10, 5.80 to 6.00, 5.80 to 5.90, 5.90 to 6.99, 5.90 to 6.90, 5.90 to6.80, 5.90 to 6.70, 5.90 to 6.60, 5.90 to 6.50, 5.90 to 6.40, 5.90 to6.30, 5.90 to 6.20, 5.90 to 6.10, 5.90 to 6.00, 6.00 to 6.99, 6.00 to6.90, 6.00 to 6.80, 6.00 to 6.70, 6.00 to 6.60, 6.00 to 6.50, 6.00 to6.40, 6.00 to 6.30, 6.00 to 6.20, 6.00 to 6.10, 6.10 to 6.99, 6.10 to6.90, 6.10 to 6.80, 6.10 to 6.70, 6.10 to 6.60, 6.10 to 6.50, 6.10 to6.40, 6.10 to 6.30, 6.0 to 6.20, 6.20 to 6.99, 6.20 to 6.90, 6.20 to6.80, 6.20 to 6.70, 6.20 to 6.60, 6.20 to 6.50, 6.20 to 6.40, 6.20 to6.30, 6.30 to 6.99, 6.30 to 6.90, 6.30 to 6.80, 6.30 to 6.70, 6.30 to6.60, 6.30 to 6.50, 6.30 to 6.40, 6.40 to 6.99, 6.40 to 6.90, 6.40 to6.80, 6.40 to 6.70, 6.40 to 6.60, 6.40 to 6.50, 6.50 to 6.99, 6.50 to6.90, 6.50 to 6.80, 6.50 to 6.70, 6.50 to 6.60, 6.60 to 6.99, 6.60 to6.90, 6.60 to 6.80, 6.60 to 6.70, 6.70 to 6.99, 6.70 to 6.90, 6.70 to6.80, 6.80 to 6.99, 6.80 to 6.90, or 6.90 to 6.99.

In certain embodiments, the reference pH value is from 7.0 to 7.2. Insome embodiments, the reference pH value is from 7.00 to 7.20, 7.00 to7.15, 7.00 to 7.10, 7.00 to 7.05, 7.05 to 7.20, 7.05 to 7.15, 7.05 to7.10, 7.10 to 7.20, 7.10 to 7.15, or 7.15 to 7.20.

In some embodiments, the increased labile proton exchange rate isgreater than 200 exchanges/second. In some embodiments, the increasedlabile proton exchange rate is from 201 to 1000 exchanges/second. Insome embodiments, the increased labile proton exchange rate is from 201to 1000, 201 to 950, 201 to 900, 201 to 850, 201 to 800, 201 to 750, 201to 700, 201 to 650, 201 to 600, 201 to 550, 201 to 500, 201 to 450, 201to 400, 201 to 350, 201 to 300, 201 to 250, 250 to 1000, 250 to 950, 250to 900, 250 to 850, 250 to 800, 250 to 750, 250 to 700, 250 to 650, 250to 600, 250 to 550, 250 to 500, 250 to 450, 250 to 400, 250 to 350, 250to 300, 300 to 1000, 300 to 950, 300 to 900, 300 to 850, 300 to 800, 300to 750, 300 to 700, 300 to 650, 300 to 600, 300 to 550, 300 to 500, 300to 450, 300 to 400, 300 to 350, 350 to 1000, 350 to 950, 350 to 900, 350to 850, 350 to 800, 350 to 750, 350 to 700, 350 to 650, 350 to 600, 350to 550, 350 to 500, 350 to 450, 350 to 400, 400 to 1000, 400 to 950, 400to 900, 400 to 850, 400 to 800, 400 to 750, 400 to 700, 400 to 650, 400to 600, 400 to 550, 400 to 500, 400 to 450, 450 to 1000, 450 to 950, 450to 900, 450 to 850, 450 to 800, 450 to 750, 450 to 700, 450 to 650, 450to 600, 450 to 550, 450 to 500, 500 to 1000, 500 to 950, 500 to 900, 500to 850, 500 to 800, 500 to 750, 500 to 700, 500 to 650, 500 to 600, 500to 550, 550 to 1000, 550 to 950, 550 to 900, 550 to 850, 550 to 800, 550to 750, 550 to 700, 550 to 650, 550 to 600, 600 to 1000, 600 to 950, 600to 900, 600 to 850, 600 to 800, 600 to 750, 600 to 700, 600 to 650, 650to 1000, 650 to 950, 650 to 900, 650 to 850, 650 to 800, 650 to 750, 650to 700, 700 to 1000, 700 to 950, 700 to 900, 700 to 850, 700 to 800, 700to 750, 750 to 1000, 750 to 950, 750 to 900, 750 to 850, 750 to 800, 800to 1000, 800 to 950, 800 to 900, 800 to 850, 850 to 1000, 850 to 950,850 to 900, 900 to 1000, 900 to 950, or 950 to 1000 exchanges/second.

In some embodiments, the reference value is a reference labile protonexchange rate, wherein the reference labile proton exchange rate is from100 to 200 exchanges/second. In some embodiments, the reference labileproton exchange rate is from 100 to 200, 100 to 190, 100 to 180, 100 to170, 100 to 160, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 100 to110, 110 to 200, 110 to 190, 110 to 180, 110 to 170, 110 to 160, 110 to150, 110 to 140, 110 to 130, 110 to 120, 120 to 200, 120 to 190, 120 to180, 120 to 170, 120 to 160, 120 to 150, 120 to 140, 120 to 130, 130 to200, 130 to 190, 130 to 180, 130 to 170, 130 to 160, 130 to 150, 130 to140, 140 to 200, 140 to 190, 140 to 180, 140 to 170, 140 to 160, 140 to150, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 150 to 160, 160 to200, 160 to 190, 160 to 180, 160 to 170, 170 to 200, 170 to 190, 170 to180, 180 to 200, 180 to 190, or 190 to 200 exchanges/second.

In some embodiments, the reference value is obtained from a normalsubject that does not have a condition. In some embodiments, thereference value is obtained from a control subject, wherein the controlsubject does not have a condition. In some embodiments, the referencevalue is obtained from the subject, wherein the subject does not have acondition. In some embodiments, the reference value is obtained from thesubject before the subject is treated for a condition. In someembodiments, the reference value is obtained from a subject that hasbeen treated for a condition. In some embodiments, the reference valueis obtained from the subject at an earlier time point (earlier point intime).

In some embodiments, the imaged region of the subject's body includesthe subject's spine or vertebral column or backbone or a section orcomponent thereof. In some embodiments, the imaged region of thesubject's body includes a joint or an intervertebral disc. In someembodiments, the imaged region of the subject's body is one or morejoints. In some embodiments, the imaged region of the subject's body isone or more intervertebral discs.

In some embodiments, the condition may include, but is in no way limitedto, intervertebral disc degeneration, discogenic pain, osteoarthritis,rheumatoid arthritis, an articular cartilage injury, temporomandibulardisc degeneration and combinations thereof. In some embodiments thecondition is selected from intervertebral disc degeneration, discogenicpain, discogenic low back pain, chronic low back pain, low back pain,back pain, chronic back pain, progressive intervertebral discdegeneration, osteoarthritis, rheumatoid arthritis, an articularcartilage injury, temporomandibular disc degeneration and combinationsthereof. In certain embodiments, the condition is discogenic pain. Incertain embodiments, the condition is discogenic low back pain. Incertain embodiments, the condition is intervertebral disc degeneration.In some embodiments, the condition is a disease condition. In someembodiments, the condition is a medical condition. In some embodiments,the condition is a disorder. In some embodiments, the condition is adisease. In some embodiments, the condition is intervertebral discdegeneration. In some embodiments, the condition is osteoarthritis. Insome embodiments, the condition is rheumatoid arthritis. In someembodiments, the condition is an articular cartilage injury. In someembodiments, the condition is temporomandibular disc degeneration. Insome embodiments, the condition is chronic low back pain. In someembodiments, the condition is low back pain. In some embodiments, thecondition is back pain. In some embodiments, the condition is chronicback pain. In some embodiments, the condition is progressiveintervertebral disc degeneration. In some embodiments, the condition isa painful condition.

In certain embodiments, the method further includes determining that anorigin of the subject's pain associated with the condition is within theregion of the subject's body where one or more abnormal physiologicalstates is detected.

In some embodiments, the magnetic resonance imaging technique is achemical exchange saturation transfer (CEST) sequence. In someembodiments, the magnetic resonance imaging technique is a quantitativechemical exchange saturation transfer sequence (qCEST). In someembodiments, the region is imaged using a chemical exchange saturationtransfer (CEST) sequence. In some embodiments the region is imaged usinga quantitative chemical exchange saturation transfer sequence (qCEST).In some embodiments, the region is imaged using a two dimension (2D)reduced field-of-view (rFOV) turbo spin echo (TSE) chemical exchangesaturation transfer (CEST) sequence. In some embodiments, the region isimaged using a two dimension (2D) reduced field-of-view (rFOV) turbospin echo (TSE) quantitative chemical exchange saturation transfer(qCEST) sequence. In some embodiments, alternative CEST sequences may beused to measure the aforementioned physiological biomarkers. In someembodiments, alternative qCEST sequences may be used to measure theaforementioned physiological biomarkers. In some embodiments, thequantitative chemical exchange saturation transfer (qCEST) sequence is atwo dimension (2D) quantitative chemical exchange saturation transfer(qCEST) sequence. In some embodiments, the quantitative chemicalexchange saturation transfer (qCEST) sequence is a three dimension (3D)quantitative chemical exchange saturation transfer (qCEST) sequence.

In various embodiments, the imaging of a region of the subject's bodywith a magnetic resonance imaging (MRI) scanner is performed in vivo. Invarious embodiments, the magnetic resonance images from the subject areobtained in vivo.

The readout of the CEST sequences may include, but are in no way limitedto, gradient echo (GRE), echo planar imaging (EPI), gradient and spinecho (GRASE) and balanced steady-state free precession (SSFP). Thereadout of the qCEST sequences may include, but are in no way limitedto, gradient echo (GRE), echo planar imaging (EPI), gradient and spinecho (GRASE) and balanced steady-state free precession (SSFP). Incertain embodiments, the method further includes diagnosing the subjectwith a condition characterized by pain and/or tissue degeneration, ifthe physiological biomarkers detected from imaging indicate one or moreabnormal physiological states within the imaged region.

In some embodiments, the MRI scanner is a 7.0T scanner. In certainembodiments, the MRI scanner is a 3.0T MRI scanner. In some embodiments,the MRI scanner is a 1.5T MRI scanner.

In certain embodiments, the imaging is performed by using reducedfield-of-view (rFOV) excitation. In other embodiments, rFOV excitationis not used. In some embodiments, a slice thickness for the MRI scan isselected to be small enough to avoid fat signal interference. In certainembodiments, CEST MRI imaging parameters include: TR/TE =10500/10 ms, 2averages, single shot. In certain embodiments, qCEST MRI imagingparameters include: TR/TE=10500/10 ms, 2 averages, single shot. Incertain embodiments, CEST MRI imaging parameters include: TR/TE 1/410,500/l Oms, 2 averages, single shot. In certain embodiments, qCEST MRIimaging parameters include: TR/TE 1/4 10,500/10 ms, 2 averages, singleshot. In some embodiments TRITE=7000-16000/7-l5 ms. In certainembodiments, for each IVD, images are acquired in the axial plane with aslice thickness of 3 mm, field of view (FOV) of 100×40 mm² and spatialresolution of 0.8×0.8 mm². In some embodiments, slice thickness is 2-4mm, FOV is 80-160×40-160 mm², and spatial resolution is 0.6-1 mm². Incertain embodiments, for each IVD, images are acquired in the axialplane with a slice thickness of 3 mm, field of view of 140×40 mm² andspatial resolution of 1.1×1.1 mm². In certain embodiments, the CESTsaturation module utilized in the imaging consists of 39 Gaussian-shapedpulses, with a duration t_(p)=80 ms for each pulse and an interpulsedelay t_(d)=80 ms (duty cycle=50%, total saturation duration T₅=6240 ms)at saturation flip angle 900°, 1500°, 2100° and 3000° (B₁amplitudes=flip angle/(yt_(p))=0.73μT, 1.22 μl, 1.71μT and 2.45μT;Gaussian saturation pulse parameters c1=0.50, c2=0.59). In certainembodiments, the qCEST saturation module utilized in the imagingconsists of 39 Gaussian-shaped pulses, with a duration t_(p)=80 ms foreach pulse and an interpulse delay t_(d)=80 ms (duty cycle=50%, totalsaturation duration T_(s)=6240 ms) at saturation flip angle 900°, 1500°,2100° and 3000° (B₁ amplitudes=flip angle/(yt_(p))=0.73μT, 1.22μT,1.71μT and 2.45μT; Gaussian saturation pulse parameters c1=0.50,c2=0.59). In some embodiments, the CEST saturation module may have totalsaturation Ts=4000-8000 ms at saturation flip angle ranging from 600 to3000°. In some embodiments, the qCEST saturation module may have totalsaturation Ts=4000-8000 ms at saturation flip angle ranging from 600 to3000°.

In certain embodiments, the Z-spectrum are acquired with differentsaturation frequencies, including but not limited to ±1.6, ±1.3, ±1.0,±0.7, and ±0.4 ppm. In some embodiments, the scan time of the CESTexperiment for each IVD was about 40 minutes. In some embodiments, thescan time of the qCEST experiment for each IVD was about 40 minutes.

As used herein, a physiological biomarker refers to, for example a pHvalue, a labile proton exchange rate (k_(SW)) between a solute pool anda water pool, or a labile proton ratio (f_(r)) between a solute pool anda water pool. The terms physiological biomarker and metabolic biomarkerhave the same meaning and are used interchangeably herein.

Systems and Computers

In various embodiments, the invention teaches a non-transitorycomputer-readable medium having computer-readable instructions forcausing one or more processors of a magnetic resonance imaging (MRI)machine to execute a method that includes applying an MRI pulse sequenceto a volume of interest (VOI) in a subject, wherein the VOI includes ajoint or an intervertebral disc (IVD), or a portion thereof; acquiringmagnetic resonance data from the volume of interest (VOI) in thesubject; and measuring, based on the magnetic resonance data acquired,one or more physiological biomarkers within the imaged region, wherein(a) the physiological biomarkers include a labile proton ratio (f_(r))between a solute pool and a water pool and/or an exchange rate (k_(SW))between the solute pool and the water pool, for example as describedherein in greater detail in the “Examples” section. In certainembodiments, the pulse sequence is a two dimension (2D) reduced field ofview (rFOV) turbo spin echo (TSE) chemical exchange saturation transfer(CEST) sequence. In certain embodiments, the pulse sequence is a twodimension (2D) reduced field of view (rFOV) turbo spin echo (TSE)quantitative chemical exchange saturation transfer (qCEST) sequence. Insome embodiments, the MRI scanner is a 7.0T scanner. In certainembodiments, the MRI scanner is a 3.0T MRI scanner. In some embodiments,the MRI scanner is a 1.5T MRI scanner.

In various embodiments, the invention teaches a magnetic resonanceimaging system that includes a magnet operable to provide a magneticfield; a transmitter operable to transmit to a region within themagnetic field; a receiver operable to receive a magnetic resonancesignal from the region; one or more processor operable to control thetransmitter and the receiver; and a non-transitory computer-readablemedium having computer-readable instructions for causing one or moreprocessor of the magnetic resonance imaging (MRI) system to execute amethod that includes: applying a pulse sequence described herein to avolume of interest (VOI) in a subject, wherein the VOI includes a jointor an intervertebral disc (IVD) or a portion thereof; acquiring magneticresonance data from the volume of interest (VOI) in the subject; andmeasuring, based on the magnetic resonance data acquired, one or morephysiological biomarkers within the imaged region, wherein (a) thephysiological biomarkers include a labile proton ratio (f_(r)) between asolute pool and a water pool and/or an exchange rate (k_(SW) ) betweenthe solute pool and the water pool.

One of skill in the art would readily appreciate that a number ofdifferent types of imaging systems could be used to perform theinventive methods described herein. Merely by way of example, theimaging systems described in the examples could be used. FIG. 5 alsodepicts a view of a system 100 that can be used to accomplish theinventive methods. System 100 includes hardware and computer 107.Hardware includes magnet 102, transmitter 103, receiver 104, andgradient 105, all of which are in communication with processor 101.Magnet 102 can include a permanent magnet, a superconducting magnet, orother type of magnet. Transmitter 103 along with receiver 104, are partof the RF system. Transmitter 103 can represent a radio frequencytransmitter, a power amplifier, and an antenna (or coil). Receiver 104,as denoted in FIG. 5 , can represent a receiver antenna (or coil) and anamplifier. In the example shown, transmitter 103 and receiver 104 areseparately represented, however, in one example, transmitter 103 andreceiver 104 can share a common coil. The hardware includes gradient105. Gradient 105 can represent one or more coils used to apply agradient for localization.

Processor 101, in communication with various elements of the hardware,includes one or more processors configured to implement a set ofinstructions corresponding to any of the methods disclosed herein.Processor 101 can be configured to implement a set of instructions(stored in memory of the hardware or sub-system 108) to provide RFexcitation and gradients and receive magnetic resonance data from avolume of interest. Sub-system 108 can include hardware and softwarecapable of facilitating the processing of data generated by thehardware, in conjunction with, or as a substitute for, the processingassociated with image reconstruction that is normally handled byprocessor 101 in an MRI machine. One of skill in the art would readilyappreciate that certain components of the imaging systems describedherein, including the processor 101 and/or sub-system 108, are used toexecute instructions embedded on a computer-readable medium to implementthe inventive data acquisition, image reconstruction, and physiologicalbiomarker evaluation methods described herein.

In some embodiments, computer 107 is operably coupled to the hardwareand sub system 108. Computer 107 can include one or more of a desktopcomputer, a workstation, a server, or a laptop computer. In one example,computer 107 is user-operable and includes a display, a printer, anetwork interface or other hardware to enable an operator to controloperation of the system 100.

In some embodiments, the invention includes using any of the methods orsystems described herein to diagnose a subject with the presence orabsence of a disease, disorder, condition, disease condition, or medicalcondition, including, but in no way limited to, back pain, one or moredegenerate discs, joint pain, one or more degenerate joints or the like,based upon the images acquired.

In various embodiments, the invention teaches a system configured toperform the methods described herein, wherein the system includes amagnetic resonance imaging device (including but in no way limited toany type of magnetic resonance imaging device described herein or in anyreference cited herein) operably connected to (through physical orelectronic communication) a computing device. The computing device mayinclude, but is in no way limited to, a desktop computer, a laptopcomputer, or handheld computing device with sufficient computingcapabilities to perform the methods described herein. In someembodiments, the computing device is specifically configured to performthe steps of one or more of the methods for image analysis set forthherein.

In accordance with the present invention, a “communication link,” asused in this disclosure, means a wired and/or wireless medium thatconveys data or information between at least two points. The wired orwireless medium may include, for example, a metallic conductor link, aradio frequency (RF) communication link, an Infrared (IR) communicationlink, an optical communication link, or the like, without limitation.The RF communication link may include, for example, WiFi, WiMAX, IEEE802.11, DECT, OG, 1G, 2G, 3G or 4G cellular standards, Bluetooth, andthe like.

Computers and computing devices typically include a variety of media,which can include computer-readable storage media and/or communicationsmedia, in which these two terms are used herein differently from oneanother as follows.

Computer-readable storage media can be any available storage media thatcan be accessed by the computer, is typically of a non-transitorynature, and can include both volatile and nonvolatile media, removableand non-removable media. By way of example, and not limitation,computer-readable storage media can be implemented in connection withany method or technology for storage of information such ascomputer-readable instructions, program modules, structured data, orunstructured data. Computer-readable storage media can include, but arenot limited to, RAM, ROM, EEPROM, flash memory or other memorytechnology, CD-ROM, digital versatile disk (DVD) or other optical diskstorage, magnetic cassettes, magnetic tape, magnetic disk storage orother magnetic storage devices, or other tangible and/or non-transitorymedia which can be used to store desired information. Computer-readablestorage media can be accessed by one or more local or remote computingdevices, e.g., via access requests, queries or other data retrievalprotocols, for a variety of operations with respect to the informationstored by the medium.

On the other hand, communications media typically embodycomputer-readable instructions, data structures, program modules orother structured or unstructured data in a data signal that can betransitory such as a modulated data signal, e.g., a carrier wave orother transport mechanism, and includes any information delivery ortransport media. The term “modulated data signal” or signals refers to asignal that has one or more of its characteristics set or changed insuch a manner as to encode information in one or more signals. By way ofexample, and not limitation, communication media include wired media,such as a wired network or direct-wired connection, and wireless mediasuch as acoustic, RF, infrared and other wireless media.

Stored on any one or on a combination of computer readable media, theexemplary embodiments of the present disclosure may include software forcontrolling the devices and subsystems of the exemplary embodiments, fordriving the devices and subsystems of the exemplary embodiments, forenabling the devices and subsystems of the exemplary embodiments tointeract with a human user, and the like. Such software can include, butis not limited to, device drivers, firmware, operating systems,development tools, applications software, database management software,and the like. Computer code devices of the exemplary embodiments caninclude any suitable interpretable or executable code mechanism,including but not limited to scripts, interpretable programs, dynamiclink libraries (DLLs), Java classes and applets, complete executableprograms, and the like. Moreover, processing capabilities may bedistributed across multiple processors for better performance,reliability, cost, or other benefits.

To provide aspects of the present disclosure, embodiments may employ anynumber of programmable processing devices that execute software orstored instructions. Physical processors and/or machines employed byembodiments of the present disclosure for any processing or evaluationmay include one or more networked (Internet, cloud, WAN, LAN, satellite,wired or wireless (RF, cellular, WiFi, Bluetooth, etc.)) ornon-networked general purpose computer systems, microprocessors, filedprogrammable gate arrays (FPGAs), digital signal processors (DSPs),micro-controllers, smart devices (e.g., smart phones), computer tablets,handheld computers, and the like, programmed according to the teachingsof the exemplary embodiments. In addition, the devices and subsystems ofthe exemplary embodiments can be implemented by the preparation ofapplication-specific integrated circuits (ASICs) or by interconnectingan appropriate network of conventional component circuits. Thus, theexemplary embodiments are not limited to any specific combination ofhardware circuitry and/or software.

Some embodiments of the present invention can be defined as any of thefollowing numbered paragraphs:

-   1. A method, comprising:    -   imaging a region of a subject's body with a magnetic resonance        imaging (MRI) scanner; and    -   measuring one or more physiological biomarkers within the imaged        region, wherein (a) the physiological biomarkers comprise a        labile proton ratio (f_(r)) between a solute pool and a water        pool and/or an exchange rate (k_(SW)) between the solute pool        and the water pool.-   2. The method of paragraph 1, wherein the region is imaged using a    chemical exchange saturation transfer (CEST) sequence.-   3. The method of paragraph 1, wherein the region is imaged using a    two dimension (2D) reduced field-of-view (rFOV) turbo spin echo    (TSE) chemical exchange saturation transfer (CEST) sequence.-   4. The method of paragraph 1, further comprising diagnosing the    subject with a condition characterized by pain and/or tissue    degeneration if the physiological biomarkers detected from imaging    indicate one or more abnormal physiological states within the imaged    region, said abnormal physiological states selected from the group    consisting of low pH and/or low GAG concentration compared to a    normal subject without the condition.-   5. The method of paragraph 1 or 2, wherein the imaged region of the    subject's body comprises a joint or an intervertebral disc.-   6. The method of paragraph 2 or 3, wherein the condition is selected    from the group consisting of: intervertebral disc degeneration,    discogenic pain, osteoarthritis, rheumatoid arthritis, an articular    cartilage injury, temporomandibular disc degeneration and    combinations thereof.-   7. The method of paragraph 3 or 4, further comprising determining    that an origin of the subject's pain associated with the condition    is within the region of the subject's body where the abnormal    physiological state is detected.-   8. The method of paragraph 1, wherein the MRI scanner is a 3.0T MRI    scanner.-   9. The method of paragraph 1, wherein the MRI scanner is a 1.5T MRI    scanner.-   10. The method of paragraph 1, wherein the MRI scanner is a 7.0T MRI    scanner.-   11. The method of paragraph 1, wherein the imaging is performed by    using reduced field-of-view (rFOV) excitation.-   12. The method of paragraph 1, wherein a slice thickness for the MRI    scan is selected to be small enough to avoid fat signal    interference.-   13. A non-transitory computer-readable medium having    computer-readable instructions for causing one or more processors of    a magnetic resonance imaging (MRI) machine to execute a method,    comprising:    -   applying an MRI pulse sequence to a volume of interest (VOL) in        a subject,    -   wherein the VOI comprises a joint or an intervertebral disc        (IVD) or a portion thereof;    -   acquiring magnetic resonance data from the volume of interest        (VOL) in the subject; and    -   measuring, based on the magnetic resonance data acquired, one or        more physiological biomarkers within the imaged region,        wherein (a) the physiological biomarkers comprise a labile        proton ratio (f_(r)) between a solute pool and a water pool        and/or an exchange rate (k_(sμ).) between the solute pool and        the water pool.-   14. The non-transitory computer-readable medium of paragraph 13,    wherein the pulse sequence is a two dimension (2D) reduced field of    view (rFOV) turbo spin echo (TSE) chemical exchange saturation    transfer (CEST) sequence.-   15. The non-transitory computer-readable medium of paragraph 13,    wherein the MRI scanner is a 3.0T MRI scanner.-   16. The non-transitory computer-readable medium of paragraph 13,    wherein the MRI scanner is a 1.5T MRI scanner.-   17. A magnetic resonance imaging system, comprising:    -   a magnet operable to provide a magnetic field;    -   a transmitter operable to transmit to a region within the        magnetic field;    -   a receiver operable to receive a magnetic resonance signal from        the region;    -   one or more processor operable to control the transmitter and        the receiver; and    -   a non-transitory computer-readable medium having        computer-readable instructions for causing one or more processor        of the magnetic resonance imaging (MRI) system to execute a        method, comprising:        -   applying a pulse sequence to a volume of interest (VOI) in a            subject, wherein the VOI comprises a joint or an            intervertebral disc (IVD) or a portion thereof;        -   acquiring magnetic resonance data from the volume of            interest (VOI) in the subject; and        -   measuring, based on the magnetic resonance data acquired,            one or more physiological biomarkers within the imaged            region, wherein (a) the physiological biomarkers comprise a            labile proton ratio (f_(r)) between a solute pool and a            water pool and/or an exchange rate (k_(SW)) between the            solute pool and the water pool.

Some embodiments of the present invention can be defined as any of thefollowing numbered paragraphs:

-   1. A method for diagnosing a condition in a subject, the method    comprising:    -   imaging a region of a subject's body with a magnetic resonance        imaging (MR1) scanner, wherein the region is imaged using a        quantitative chemical exchange saturation transfer (qCEST)        sequence;    -   measuring one or more physiological biomarkers within the imaged        region, wherein the physiological biomarkers comprise a labile        proton exchange rate (k) between a solute pool and a water pool;        and    -   determining that the subject has the condition if the labile        proton exchange rate is increased relative to a reference value.-   2. The method of paragraph 1, wherein the increased labile proton    exchange rate is correlated to a low pH value.-   3. The method of paragraph 1, wherein the increased labile proton    exchange rate is greater than 200 exchanges/second.-   4. The method of paragraph 1, wherein the increased labile proton    exchange rate is from 201 to 1000 exchanges/second.-   5. The method of paragraph 2, wherein the low pH value is from 5.6    to 6.99.-   6. The method of paragraph 1, wherein the reference value is a    reference labile proton exchange rate, wherein the reference labile    proton exchange rate is from 100 to 200 exchanges/second.-   7. The method of paragraph 6, wherein the reference labile proton    exchange rate is correlated to a reference pH value.-   8. The method of paragraph 7, wherein the reference pH value is from    7.0 to 7.2.-   9. The method of paragraph 1, wherein the wherein the condition is    selected from intervertebral disc degeneration, discogenic pain,    discogenic low back pain, chronic low back pain, low back pain, back    pain, chronic back pain, progressive intervertebral disc    degeneration, osteoarthritis, rheumatoid arthritis, an articular    cartilage injury, temporomandibular disc degeneration and    combinations thereof.-   10. The method of paragraph 1, wherein the imaged region of the    subject's body comprises a joint or an intervertebral disc.-   11. The method of paragraph 1, wherein the condition is a painful    condition.-   12. The method of paragraph 1, wherein the increased labile proton    exchange rate is correlated with an upregulation of one or more    pain-related factors in the subject.-   13. The method of paragraph 12, wherein the one or more pain-related    factors are selected from bradykinin receptor B1 (BDKRB1),    calcitonin gene-related peptide (CGRP), and    catechol-0-methyltransferase (COMT).-   14. The method of paragraph 1, wherein the increased labile proton    exchange rate is correlated with an upregulation of one or more    inflammation-related factors in the subject.-   15. The method of paragraph 14, wherein the inflammation-related    factor is interleukin-6 (IL-6).-   16. The method of paragraph 1, wherein the increased labile proton    exchange rate is correlated with an upregulation of one or more    neurogenic factors in the subject.-   17. The method of paragraph 16, wherein the neurogenic factor is    brain-derived neurotrophic factor (BDNF) or nerve growth factor    (NGF).-   18. The method of paragraph 1, wherein the quantitative chemical    exchange saturation transfer (qCEST) sequence is a two dimension    (2D) quantitative chemical exchange saturation transfer (qCEST)    sequence.-   19. The method of paragraph 1, wherein the quantitative chemical    exchange saturation transfer (qCEST) sequence is a three dimension    (3D) quantitative chemical exchange saturation transfer (qCEST)    sequence.-   20. The method of paragraph 1, wherein the MRI scanner is a 3.0T MRI    scanner.-   21. The method of paragraph 1, wherein the MRI scanner is a 1.5T MRI    scanner.-   22. The method of paragraph 1, wherein the MRI scanner is a 7.0T MRI    scanner.-   23. The method of paragraph 1, further comprising determining that    an origin of the subject's condition is within the imaged region of    the subject's body where the physiological biomarker was measured.-   24. The method of paragraph 2, wherein the low pH value is    indicative of the subject having the condition.-   25. The method of claim 1, further comprising selecting one or more    treatments for the subject if the condition is determined.-   26. The method of claim 1, further comprising treating the subject    with one or more treatments if the condition is determined.

Various embodiments of the present invention are described in theensuing examples. The examples are intended to be illustrative and in noway restrictive.

Examples

The following examples are not intended to limit the scope of the claimsto the invention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods which occur tothe skilled artisan are intended to fall within the scope of the presentinvention.

The invention will be further explained by the following examples, whichare intended to be purely exemplary of the invention, and should not beconsidered as limiting the invention in any way. The following examplesare provided to better illustrate the claimed invention and are not tobe interpreted as limiting the scope of the invention. To the extentthat specific materials are mentioned, it is merely for purposes ofillustration and is not intended to limit the invention. One skilled inthe art may develop equivalent means or reactants without the exerciseof inventive capacity and without departing from the scope of theinvention.

Example 1 Theory

By way of additional background, previous studies have focused on thetwo-pool exchange model using Bloch-McConnell equations, describing theproton exchange between pool ‘w’ (water pool) and pool ‘s’ (solutepool). In this two-pool system, f_(r) refers to the labile proton ratioM_(0s)/M_(0w) and k_(SW) refers to the exchange rate between solute pooland water pool. R_(1w), R_(2w), R_(1s), and R_(2s) are longitudinal andtransverse relaxation rates for water protons and solute protons,respectively.

The conventional CEST asymmetry analysis takes direct difference betweenthe label scan (at the resonant frequency of the solute pool) andreference scan (at the opposite frequency with respect to water). It canbe defined as CESTR=Z_(label)-Z_(ref), where Z_(tabel) and Z_(ref) arethe normalized signal intensity or Z-spectrum for the label scan andreference scan.

Recent studies have simplified the inverse CEST difference (CESTR,w) as

$\begin{matrix}{\frac{1}{{CESTR}_{ind}} = {\frac{1}{\frac{1}{Z_{label}} - \frac{1}{Z_{ref}}} \approx {\frac{R_{1w}}{f_{r} \cdot k_{sw}} + {\frac{k_{sw} \cdot \left( {R_{2s} + k_{sw}} \right) \cdot R_{1w}}{f_{r} \cdot k_{sw}}\frac{1}{\omega_{1}^{2}}}}}} & \lbrack 1\rbrack\end{matrix}$

where Z_(label)and Z_(ref) are the normalized signal intensity orZ-spectrum for the label scan (at the resonant frequency of the solutepool) and reference scan (at the opposite frequency with respect towater). ω₁ is the RF irradiation amplitude.

Eq. 1 is only valid for cw CEST saturation. When pulsed saturation isapplied in CEST experiments, Eq. 1 can be written as

$\begin{matrix}{\frac{1}{{CESTR}_{ind}} \approx {\frac{R_{1w}}{D{C \cdot f_{r} \cdot k_{sw} \cdot c_{1}}} + {\frac{k_{sw} \cdot \left( {R_{2s} + k_{sw}} \right) \cdot R_{1w} \cdot c_{2}^{2}}{D{C \cdot f_{r} \cdot k_{sw} \cdot c_{1}}}\frac{1}{\omega_{1}^{2}}}}} & \lbrack 2\rbrack\end{matrix}$

where DC stands for duty cycle; c₁ and c₂ describe the shape of Gaussiansaturation pulses

$\left( {{c_{1} = {\sigma\sqrt{2\pi}/t_{p}}},{{c_{2} = {c_{1}\sqrt{\sqrt{2}}}};}} \right.$

σ and t_(p)are the width and length of the Gaussian pulse). Note ω₁ hereis defined as the average RF irradiation amplitude of one Gaussianpulse, i.e., ω₁=flip angle/pulse duration.

In this expression, 1/CESTR_(ind) is described as a linear function of1/ω₁ ². By measuring CESTR_(ind) with different RF irradiationamplitude, the slope m and intercut n can be calculated, and eventuallyk_(SW) and f_(r) can be estimated.

$\begin{matrix}{k_{sw} = \frac{\sqrt[2]{R_{2s}^{2} + \frac{4m}{n \cdot c_{2}^{2}}} - R_{2s}}{2}} & \left\lbrack {{Eq}.3} \right\rbrack\end{matrix}$ $\begin{matrix}{f_{r} = \frac{R_{1w}}{{k_{sw} \cdot n \cdot c_{1} \cdot D}C}} & \left\lbrack {{Eq}.4} \right\rbrack\end{matrix}$

R_(1w) can be measured using T₁ mapping techniques. R_(2s) of GAG isestimated to be 200 s⁻¹(See Lee J-S, Xia D, Jerschow A, Regatte RR. Invitro study of endogenous CEST agents at 3 T and 7 T. Contrast Media MolImaging 2016; 11:4-14. doi: 10.1002/cmmi.1652, which is herebyincorporated herein by reference in its entirety as though fully setforth). Note Eq. 1 is a simplified expression that describes the steadystate of CEST experiments. When performing qCEST experiments, RFsaturation pulses need to be long enough to ensure the steady state isreached. The simplification only holds for dilute CEST agents undergoingslow and intermediate chemical exchange.

Methods Phantom

Two sets of phantoms containing GAG prepared from chondroitin sulphate A(Aldrich-Sigma, St. Louis, Mo.) and phosphate buffer solution (PBS) withvarying pH values and concentrations were prepared. For the pH set, theGAG concentration was fixed at 60 mM and pH was titrated to 5.8, 6.1,6.4, 6.7 and 7.0. For the concentration phantom, various GAGconcentrations (100 mM, 80 mM, 60 mM, 40 mM and 20 mM) were used and thepH titrated to 7.0. The solution was then transferred to 15 mL tubes.These ten tubes were put in a phantom holder filled with water.

In vitro MRI experiments

Imaging experiments were performed at room temperature on a 3.0 Teslaclinical scanner (Magnetom Verio; Siemens Healthcare, Erlangen,Germany). All images were acquired with a slice thickness of 8 mm, filedof view of 160×160 mm² and imaging matrix of 128×128. CEST MRI wasperformed with pulsed RF saturation turbo spin echo (TSE) sequence(TR/TE=16000/12 ms; 2 averages). CEST saturation module consists of 39Gaussian-shaped pulses, with a duration t_(p)=80 ms for each pulse andan interpulse delay td=80 ms (duty cycle=50%, total saturation timeTsat=6240 ms) at saturation flip angle 900°, 1500°, 2100° and 3000° (B₁amplitudes=flip angle/(yt_(t))=0.73μT, 1.22μT, 1.71μT and 2.45μT;Gaussian saturation pulse parameters c1=0.50, c2=0.59). Z-spectrum wasacquired with varying saturation frequencies (10 different saturationfrequencies) at ±1.6 ppm, ±1.3 ppm, ±1.0 ppm, ±0.7 ppm and ±0.4 ppm. B₀field was corrected using a water saturation shift referencing (WASSR)map (See Kim M, et al. Water saturation shift referencing (WASSR) forchemical exchange saturation transfer (CEST) experiments. MagneticResonance in Medicine 2009; 61:1441-1450. doi: 10.1002/mrm.21873, whichis hereby incorporated herein by reference in its entirety as thoughfully set forth). T₁-weighted MR images were acquired by an inversionrecovery TSE sequence with 10 different inversion delays (T1=50-4000 ms(i.e., TI=50, 150, 350, 700, 1050, 1400, 2000, 2500, 3000, and 4000 ms);TR/TE=6000/12 ms). T₂-weighted MR images were acquired by a TSE sequencewith varying echo delays (TE=12-399 ms (i.e., TE=12, 24, 48, 97, 205 and399 ms); TR=6000 ms).

Animal Preparation

All animal-related procedures were approved by the Institutional AnimalCare and Use Committee (IACUC) at Cedars-Sinai Medical Center. A totalof four female Yucatan minipigs (S&S Farms) were used. Following an18-hour preoperative fast, each pig was sedated with intramuscular drugs(acepromazine 0.25 mg/kg, ketamine 20 mg/kg, and atropine 0.02-0.05mg/kg), following which the animal was injected intravenously withpropofol (2 mg/kg) to induce full anesthesia. After this had beenachieved, the trachea was intubated and anesthesia was maintained using1-3.5% isoflurane inhaled via the tracheal tube for the duration of theprocedure. Following anesthesia, under fluoroscopic guidance threeMR-compatible 14G coaxial needles (Invivo, Gainesville, FL) wereinserted into the mid substance of lumbar discs L1/L2, L3/L4 and L5/L6.These lumbar discs were injected with different concentrations ofNa-Lactate (Sigma Aldrich, St. Louis, Mo.) in order to induce a gradientof pH values within the discs ranging from 5-7, as described by Melkuset al, and in accordance with pH values measured within patients'pathological discs. Following intra-discal injection, exact pH valuesinside the discs were measured using a custom-made needle-shaped tissuepH probe (Warner Instruments, LLC, Hamden, Conn.) which was insertedthrough the MR-compatible needle, shortly before the MR scan. Lumbardisc L2/L3 was also scanned as the control disc. Its pH value wasmeasured immediately after the animal was euthanized.

In vivo MRI experiments

Imaging experiments were performed on a 3.0 Tesla clinical scanner(Magnetom Verio; Siemens Healthcare, Erlangen, Germany). Animal wasplaced in right decubitus position with body array coils wrappedcentered on posterior aspect spinous process. Throughout the imagingprocedures, anesthesia was maintained with isoflurane (1-3.5%).

CEST MRI was performed using a two-dimension (2D) reduced filed-of-view(rFOV) TSE CEST sequence (TR/TE=10500/10 ms, 2 averages, single shot).rFOV can effectively suppress bowel motion artifacts and increase scanefficiency (see Liu Q et al. Reliable chemical exchange saturationtransfer imaging of human lumbar intervertebral discs usingreduced-field-of-view turbo spin echo at 3.0 T. NMR in Biomedicine 2013;26:1672-1679. doi: 10.1002/nbm.3001, which is hereby incorporated hereinby reference in its entirety as though fully set forth). For each IVD,images were acquired in the axial plane with a slice thickness of 3 mm,filed of view of 100×40 mm² and spatial resolution of 0.8×0.8 mm². CESTsaturation module consists of 39 Gaussian-shaped pulses, with a durationt_(p)=80 ms for each pulse and an interpulse delay t_(d)=80 ms (dutycycle=50%, total saturation duration T₅=6240 ms) at saturation flipangle 900°, 1500°, 2100° and 3000° (B₁ amplitudes=flip angle/(y^(t)_(p))=0.73μT, 1.22μT, 1.71μT and 2.45μT; Gaussian saturation pulseparameters c1=0.50, c2=0.59). Z-spectrum was acquired with varyingsaturation frequencies (10 different saturation frequencies) at ±1.6ppm, ±1.3 ppm, ±1.0 ppm, ±0.7 ppm and ±0.4 ppm. Scan time of the CESTexperiment for each RF irradiation amplitude is −6 min. B_(o) field wascorrected using WASSR. T₁-weighted MR images were acquired by aninversion recovery TSE sequence with 7 varying T₁ (50 ms, 150 ms, 350ms, 700 ms, 1050 ms, 1400 ms and 2000 ms). Images were acquired at thesame slice position as the CEST MRI sequence (TR/TE=6000/12 ms; 1average; FOV=200×200 mm²; spatial resolution=0.8×0.8×3 mm³; scantime=−2.5 min).

Data Analysis

Post processing was performed with custom-written programs in Matlab(The Mathworks, Natick, Mass., USA). CESTR_(ind) was calculatedaccording to Eq. 1 after B_(o) correction at 1.0 ppm (Zi_(th)=Z(+1.0ppm), Z_(ref)=Z(−1.0 ppm)). Linear regression was used to perform 0-plotanalysis between 1/CESTR_(id) and 1/ω₁ ² to obtain the slope andintercut. The exchange rate k_(sw) , and labile proton ratio f, werecalculated afterwards following Eqs. [3] and [4]. These calculationswere performed pixel-by-pixel and by region of interest (ROI). The T₁maps and T₂ maps were obtained by pixel-by-pixel logarithmic fit of thesignal equation I=I₀[1-(1+)·exp(−TI/T₁)] where I is the signalintensity, TI is the inversion time and is the inversion efficiency. TheT₂ maps were obtained by fitting the signal equation I=I₀ ·exp(−TE/T₂)]where I is the signal intensity and TE is the echo time.

Results Phantom

In FIG. 1 , the relationship between 1/CESTR_(ind) and 1/ω₁ ² wereevaluated in tubes with varying GAG concentration and pH values.1/CESTR;_(ind) is the average signal within the region-of-interest (ROI)of each tube. In all tubes, 1/CESTR_(ind) can be represented as a linearfunction of 1/ω_(t) ². This experimental finding is consistent with Eq.[2].

In addition, pixel-wise mapping of chemical exchange rate k and labileproton ratio f_(r) were reconstructed, shown in FIG. 2A-FIG. 2B. It canbe seen how chemical exchange rates change as pH values vary (FIG. 2A)and how labile proton ratios change as GAG concentrations vary (FIG.2B). Quantitatively, the chemical exchange rate can be described ask_(sw)=1.5×10^(−pH+8) +252.0, R²=0.9508 (FIG. 2C). This follows an acidcatalyzed chemical exchange formula (See Englander SW, et al. Hydrogenexchange. Annu. Rev. Biochem. 1972; 41:903-924. doi:10.1146/annurev.bi.41.070172.004351, which is hereby incorporated hereinby reference in its entirety as though fully set forth). The labileproton ratio is linearly correlated with GAG concentration (FIG. 2D). Itcan be represented as f, =4.6×10⁻⁵[GAG]-4.4×10′⁵ (R²=0.9869), where[GAG] is the GAG concentration in mM. The error bars in FIG. 2 c and 2 drepresent the standard deviation of all the pixels within the ROI ofeach tube for 4, and f_(r), respectively. These experimental resultsencouraged in vivo application of qCEST technique.

Animal Studies

16 IVDs were studied in this work, 3 of which were excluded because theneedle went through both sides of the IVD and caused morphologicaldamage. The pH values of the studied 1VDs after Na-Lactate injectionranged from 5.0 to 7.2. FIG. 3A-FIG. 3C shows the anatomical images ofone representative mini-pig's lumbar IVDs and the corresponding exchangerate maps. As shown in FIG. 3A-FIG. 3C, the exchange rate was higher inthe IVDs with lower pH values. Within each disc, there was someinhomogeneity in the exchange rate map. This is because the current SNRcannot guarantee accurate measurement for a signal pixel. However, theaverage value of each IVD proved to be more reliable. This is becauseSNR will increase after averaging all pixels that are in the similar pHenvironment.

In FIG. 4A, we evaluated the relationship between 1/CESTR;_(nd) and 1/ω₁² in representative IVDs with different pH values (5.0, 5.8 and 6.7).Similar as shown in phantom studies, 1/CESTR_(ind) can be represented asa linear function of 1/ω₁ ². In FIG. 4B, the average exchange rate ofeach disc was taken, and its relationship evaluated with thecorresponding pH value, which was obtained by directly measuring theintra-discal pH value using a pH probe. The exchange rate can bedescribed by an acid catalyzed chemical exchange formula,

k_(SW) =9.2×10^(−pH+6)+196.9, R²=0.7883.

DISCUSSION

In this study, it was investigated whether qCEST analysis can be used todetect pH changes in IVDs in vivo on a 3.0 Tesla MR scanner. The phantomstudies showed that the approximations used in qCEST analysis stillholds true for molecules like GAG and the exchange rate determined fromqCEST analysis is dependent on pH levels of GAG solutions. Therelationship between the exchange rate and pH values was further studiedin the in vivo porcine spine studies. The results showed the exchangerate can be described as a function of pH using acid catalyzed protonchemical exchange formula. This is believed to be the first in vivostudy to show the validity of qCEST analysis using tissue pH meter asreference.

Previous studies have investigated the pH dependence of gagCEST. Eventhough the GAG concentration can be corrected using T_(1p), waterrelaxation parameters T₁ and T₂ still contribute to the gagCEST signal.qCEST analysis, on the other hand, has been shown to detect pH changesindependent of T₁, T₂ and concentration in numerical simulations and inphantom studies. It is a more reliable approach to measure pH changes inthe IVD, because T₁ and T₂ change significantly after disc degeneration.In this in vivo study, a relationship was established between exchangerates and pH levels, which can be potentially applied in future studiesto translate exchange rates to pH levels.

Pulsed CEST saturation pulses were used because this study was performedon a 3.0 Tesla clinical MR scanner. Pulsed qCEST analysis is even morecomplicated because of the constant changing RF irradiation amplitude.Pulsed CEST experiments normally report the irradiation power as theequivalent cw B₁ field strength. However, the proton exchange in pulsedCEST experiments is rather complicated. Simply integrating theequivalent cw B₁ field strength will cause errors in estimating theexchange rate and labile proton ratio. Meissner et al. came up with ananalytical solution for pulsed CEST experiment (See Meissner J-E, et al.Quantitative pulsed CEST-MRI using Q-plots. NMR in Biomedicine referencein its entirety as though fully set forth). This enables more accuratequantitative results of pulsed CEST experiments.

In this study, the relationship between exchange rates and pH levelswere explored in both phantom studies and in vivo animal studies.However, the results are not exactly the same. One reason is these twostudies were performed at different temperatures (−20° C. for phantomstudies and −38° C. for animal studies). Another possible reason is GAGin the IVD experiences a more complicated environment. In addition toCEST effects, magnetization transfer (MT) effects are also present inthe IVD from semi-solid components such as macromolecules, which couldaffect the qCEST analysis.

Regular CEST experiments are relatively slow, because of long TR,multiple averages, etc. In addition to that, qCEST analysis alsorequires (a) long RF saturation time (6 s in the present study) toachieve the steady state and (b) multiple CEST experiments with varyingRF irradiation amplitudes to perform the 0-plots. Compressed sensing andparallel imaging techniques can be utilized to accelerate qCESTexperiments (See Heo HY, Zhang Y, Lee DH, Jiang S, Zhao X, Zhou J.Accelerating chemical exchange saturation transfer (CEST) MRI bycombining compressed sensing and sensitivity encoding techniques.Magnetic Resonance in Medicine 2016:n/a-n/a. doi: 10.1002/mrm.26141,which is hereby incorporated herein by reference in its entirety asthough fully set forth), and therefore the implementation of thosetechniques in conjunction with the inventive systems and methods arecontemplated as within the scope of the present invention.

The manipulation of pH levels in the IVDs by injecting Na-Lactate mimicsthe degeneration condition only to a limited extent. In addition to pHchange, disc degeneration is also correlated with a loss of GAG andwater content in the nucleus pulposus. GAG loss will significantly lowerthe CEST values and the dehydration process will cause the change of MRrelaxation parameters.

CONCLUSION

The experiments reported in the present application demonstrate thefeasibility of in vivo qCEST analysis of GAG in IVDs. The experimentsreported in the present application also demonstrate that the exchangerate determined from qCEST analysis is closely correlated with pH value,and can be used to non-invasively measure pH in IVDs. qCEST techniquehas the potential to provide additional information on IVD physiologyand help gain insight into the pathogenesis of low back pain and itsunderlying degenerative processes.

Example 2.

Results Induction of IVD Degeneration

Minipigs underwent surgery during which the annulus fibrosus of fourIVDs (L1/2-L415) were punctured to induce degeneration (FIG. 6 ). Theprogress of IVD degeneration was monitored using MRI, with a cleardecrease in the intensity of the T₂-weighted signal in punctured IVDscompared to healthy IVDs (FIG. 7A). A two-fold decrease in water contentwithin the punctured IVDs was evident as soon as 2 weeks after inductionof degeneration compared to healthy porcine IVDs based on T₂-weightedmappings (p<0.0001; FIG. 7B). The water content was further reduced 10weeks after induction of degeneration compared to week 2 (p<0.05). Inaddition, a significant reduction of T₁ signal from 1.6 to 1.4 wasnoticeable at 2 weeks after induction of degeneration compared tohealthy controls (p<0.0001; FIG. 7C). Further reduction of T_(i) signalto 1.2 was measured 6 weeks after induction of degeneration (p<0.0001).T_(1p) mapping revealed two-fold reduction in signal as soon as 2 weeksafter induction of degeneration (p<0.0001; FIG. 7D). Overall, thesequantitative signals show the rapidly progressive degenerative status ofthe punctured IVDs. Histology revealed an abnormal IVD structure andcell matrix following puncture, with extensive fibrosis and formation ofcell clusters in the nucleus pulposus, typical of degenerated IVDs (FIG.7E).

MR signal correlates with infra-discal pH in degenerated HDs

In addition to measuring the degenerative status of the IVDs, qCESTsignals were acquired from the degenerative IVDs. These values werecorrelated to their correspondent pH readings that were measureddirectly from the IVDs (FIG. 8A). Strong correlation was observedbetween the qCEST signal and pH of the degenerated IVDs (R²=0.8004;p<0.0001). The available qCEST readings at weeks 2, 6 and 10 wereclassified as either healthy or degenerated and used to create an ROCcurve (area under the curve=0.813, p=0.0003) with 81.3% sensitivity and76.1% specificity (FIG. 8B). Significant pH drop from 7.2 to 6.3 wasmeasured in IVDs as soon as 2 weeks after injury (p=0.0001, FIG. 8C).This reduction in pH was maintained until week 10. In accordance withthe pH drop, the acquired qCEST signal significantly increased 2 weeksafter injury (p<0.05, FIG. 8D). Further increase in signal was observedat 6 and 10 weeks (p<0.01), demonstrating high sensitivity of the MRprotocol to small changes in pH that were not statistically significantby physical measurements.

MR signal correlates with pain-markers in degenerated IVDs

Next, we evaluated the expression of pain-related factors in thedegenerated IVDs. Harvested degenerated IVDs underwent gene expressionanalysis of RNA that was extracted from the nucleus pulposus and annulusfibrosus. Specifically, we evaluated the expression of bradykininreceptor B1 (BDKRB1), calcitonin gene-related peptide (CGRP) andcatechol-0-methyltransferase (COMT). A 10-fold increase in CGRPexpression was observed in the nucleus pulposus 6 and 10 weeks afterdegeneration (p<0.05, FIG. 9A). A 13-fold increase in BDKRB1 expressionwas observed in the annulus fibrosus 6 weeks after degeneration(p<0.05), while a non-significant decrease was observed 10 weeks afterdegeneration (p=0.1367, FIG. 9B). There was a 4-fold increase in COMTexpression in the annulus fibrosus at 2, 6 and 10 weeks afterdegeneration (p<0.01, FIG. 9C). In addition, expression of IL-6, whichis involved with inflammatory processes, were also assessed followingdegeneration. IL-6 expression analysis revealed 20-fold increase in theannulus fibrosus at 6 and 10 weeks after degeneration (p<0.05, FIG. 9D).We also evaluated the expression of brain-derived neurotrophic factor(BDNF), which is involved with nerve growth. A 22-fold increase in BDNFexpression in the annulus fibrosus was observed at 6 weeks afterdegeneration (p<0.05, FIG. 9E). Surprisingly, a non-significantdownregulation was observed in the annulus fibrosus 10 weeks afterdegeneration (p=0.0925), while a significant 18-fold increase wasobserved in the nucleus pulpusos during that time (p<0.05).Immunofluorescent staining of the nucleus pulposus with antibodiesagainst the aforementioned markers revealed increased signals andco-localizations of the studied pain markers compared to healthy IVDs(FIG. 10 ). Co-localization of COMT and IL-6 was observed within thedegenerated IVDs, linking these processes as part of the degenerativeprogression. In contrast to the gene analysis results, no expression ofBDNF was observed in the nucleus pulposus 10 weeks after degeneration.However, in some cases protein expression is not measurable inimmunofluorescent stainings, and gene expression studies are generallymore sensitive. Overall, we found that there is upregulation of severalpain, inflammatory and neurogenic factors in the degenerated IVDs.Finally, the measured qCEST signals were paired with the expressionlevels of the aforementioned markers derived from the same IVDs,resulting in strong linear correlations (p<0.0001 for all pairings; FIG.11A-FIG. 11E). Combined with our other results described herein, thisdata demonstrates that an increase in qCEST signal is correlated with anupregulation of several pain markers within the IVDs, and thereforeenables detection of painful IVDs.

DISCUSSION

Previous studies have attempted to evaluate pH as a measure fordiagnosing discogenic low back pain. Zuo et al. demonstrated thefeasibility of acquiring localized^(t)H spectra on a 3.0T scanner onintact bovine and human cadaveric IVDs to quantify lactate, which causeslow pH (J. Zuo, E. Saadat, A. Romero, K. Loo, X. Li, T. M. Link, J.

Kurhanewicz, S. Majumdar, Assessment of intervertebral disc degenerationwith magnetic resonance single-voxel spectroscopy. Magn Reson Med 62,1140-1146 (2009). However, translating this technique to in vivospectroscopy suffers from several limitations. As stated by the authors,it is difficult to differentiate lactate from the lipid peaks, becausetheir resonance frequencies are close. Another limitation is inadequatequantification of metabolites in IVDs within the collapsed space. Alater study from the same group characterized IVD in vivo byspectroscopy (J. Zuo, G. B. Joseph, X. Li, T. M. Link, S. S.

Hu, S. H. Berven, J. Kurhanewitz, S. Majumdar, In vivo intervertebraldisc characterization using magnetic resonance spectroscopy and Tlrhoimaging: association with discography and Oswestry Disability Index andShort Form-36 Health Survey. Spine (Phila Pa 1976) 37, 214 221 (2012)).A significant elevated water/proteoglycan area ratio was found in IVDswith positive discography. In vivo MRS is challenging because of lowSNR, physiological motion, and bone susceptibility induced linebroadening, making the assessment of lactate imprecise.

Another study found a non-linear dependence of the CEST effect of GAG onpH in porcine IVD specimens (G. Melkus, M. Grabau, D. C. Karampinos, S.Majumdar, Ex vivo porcine model to measure pH dependence of chemicalexchange saturation transfer effect of glycosaminoglycan in theintervertebral disc. Magn Reson Med 71, 1743-1749 (2014)). However, thestudy was performed ex vivo at 7.0T MRI and the effectiveness of themethod on clinical MR systems (1.5 or 3.0T) has not been shown.

In contrast to previous studies, in this study, we used MRI to detectdiscogenic low back pain in vivo in a minipig model of IVD degeneration.We showed that degeneration was achieved by 10 weeks following injury,as detected by MRI and histology. A significant pH drop was observedduring the degenerative process, as well as a significant increase inthe qCEST signal. These changes were detected as early as 2 weeks afterinjury. qCEST signals were well-correlated with pH measurements obtaineddirectly from the degenerated IVDs. Gene analysis revealed upregulationof several pain markers in degenerated IVDs, and this upregulation wasstrongly correlated to the increase in qCEST signal at various timepoints.

As such, in various embodiments, the present invention can beimplemented on clinical MRI systems. qCEST technique can be used toprovide additional information on IVD physiology and detect pH changesassociated with early degeneration, and thus provide early diagnosis topatients. In various embodiments, the present invention also allows forearlier interventions for IVD degeneration and prevention of chronic lowback pain.

Materials and Methods

Study design

The objective of our study was to develop a pH-level dependent MRimaging approach to diagnose low back pain. Our pre-specified hypothesiswas that pain in the degenerating IVDs is caused at least partially dueto an intra-discal acidic environment, and this pH drop can be detectednon-invasively using qCEST imaging. Nine healthy female skeletallymature Yucatan minipigs (S&S Farms; Average age 1.5 years, 35-40 kg)were included in this study. The sample size used was estimated toachieve a power of 0.8 and a=0.05 using one-way ANOVA. qCEST wasinvestigated for its capacity to detect any pH changes within the IVDs,and see whether this change can be correlated to pain markerupregulation. For this purpose, we created an IVD degeneration model ina large, clinically-relevant animal model by puncturing the annulusfibrosus with a 14 G needle, thus creating four degenerating IVDs perminipig (FIG. 6 ). Then, the minipigs went through MRI scan at 2, 6 and10 weeks after degeneration. At each time-point, three pigs wererandomly euthanized in order to directly measure the pH within the IVDusing pH meter, and the degenerated IVDs were harvested for geneexpression analysis, histology and immunofluorescence. IVD degenerationwas evaluated using imaging parameters and histology. Pain was detectedusing gene expression and immunofluorescence, and was compared qCESTmeasurements within the IVDs. Animals that developed acute proceduralcomplications such as nerve damage or signs of distress during follow-upthat compromised animal welfare were eliminated from the study.

IVD Degeneration Animal Model

All animal procedures were approved by the Cedars-Sinai Medical Centerinstitutional review board for animal experiments. The IVD degenerationmodel was created with modifications from a previously establishedmethod (O. Mizrahi, D. Sheyn, W. Tawackoli, S. Ben-David, S. Su, N. Li,A. Oh, H. Bae, D. Gazit, Z. Gazit, Nucleus pulposus degeneration altersproperties of resident progenitor cells. Spine 0.113, 803-814 (2013)).Following an 18-hour preoperative fast, each minipig was sedated usingintramuscular acepromazine (0.25 mg/kg), ketamine (20 mg/kg), andatropine (0.02-0.05 mg/kg). The animal was then administered propofol (2mg/kg) intravenously and endotracheal intubation was performed.Anesthesia was maintained using 1-3.5% inhaled isoflurane for theduration of the procedure. In order to induce IVD degeneration, a singleannular injury was performed, as it was found the most reliable andreproducible method compared to nucleus aspiration or injection ofapoptotic agents (K. S. Kim, S. T. Yoon, J. Li, J. S. Park, W. C.Hutton, Disc degeneration in the rabbit: a biochemical and radiologicalcomparison between four disc injury models. Spine (Phila Pa 1976) 30,33-37 (2005)). Under fluoroscopic guidance, a 14 G Verteport needle(Stryker, Kalamazoo, Mich.) was used to penetrate and injure the annulusfibrosus of the IVD parallel to the endplate via a posterolateralapproach. This procedure was repeated at four target levels: L1/L2,L2/L3, L3/L4 and L4/L5.

In vivo MRI

Imaging experiments were performed on a 3T clinical scanner (MagnetomVerio; Siemens Healthcare, Erlangen, Germany). Animals were placed inthe right decubitus position with body array coils centered on theposterior aspect spinous process. Throughout the imaging procedures,anesthesia was maintained with isoflurane (1%-3.5%).

CEST MRI was performed using a two-dimensional reduced field of view TSECEST sequence (TR/TE V4 10,500/10 ms, two averages, single shot). Foreach IVD, images were acquired in the axial plane with a slice thicknessof 3 mm, field of view of 140×40 mm², and spatial resolution of 1.1×1.1mm². CEST saturation module consists of 39 Gaussian-shaped pulses, witha duration t_(p)=80 ms for each pulse and an interpulse delay t_(d)=80ms (duty cycle =50%, total saturation duration T₅=6240 ms) at saturationflip angle 900, 1500, 2100, and 3000 [B₁ amplitudes=flipangle/(gt_(p))=0.73, 1.22, 1.71, and 2.45 AT; Z-spectrum was acquiredwith 10 different saturation frequencies at ±1.6, ±1.3, ±1.0, ±0.7, and±0.4 ppm. The scan time of the CEST experiment for each ND wasapproximately 40 min. The B_(o) field was corrected using a watersaturation shift referencing (WASSR) map.

T₁ mapping was performed using an inversion recovery TSE sequence withseven varying TI (50, 150, 350, 700, 1050, 1400, and 2000 ms). Otherimaging parameters are: TR/TE=6000/12 ms; FOV=280^(x)280 mm²; spatialresolution=1.1^(x)1.1×3 mm³.

T₂ mapping was performed using a TSE sequence with varying echo delays(TE=12, 25, 50, 99, 199 and 397 ms; TR=6000 ms). Other imagingparameters are: TR=6000 ms; FOV=280×280 mm²; spatialresolution=1.1×1.1×3 mm³.

T_(1p) mapping was performed using a rFOV TSE sequence with varying spinlock times (TSL=0, 10, 40 and 80 ms). The spin-lock frequency is 300 Hz.Other imaging parameters are: TR/TE=3500/9.1 ms; 1 average; FOV=140×40mm²; spatial resolution=1.1×1.1 3 mm³). Imaging data analysis wasperformed with custom-written programs in MATLAB (MathWorks, Natick,Mass., USA).

BD pH measurement

Measurements of the pH inside the IVD were done immediately followinganimals' sacrifice. The spine was surgically exposed and a custom-madeneedle-shaped tissue pH probe (Warner Instruments, Hamden, Conn., USA)was inserted to the nucleus pulposus of the injured IVDs through afine-cut incision of the annulus fibrosus.

Gene expression analysis

A quantitative RT-PCR was conducted on degenerated IVDs harvested at 2,6 and 10 weeks after degeneration. The expression of genes fromdegenerative IVDs was compared to healthy IVDs harvested from each timepoint. Total RNA was extracted from the annulus fibrosus and the nucleuspulposus by using RNeasy Mini kit (Qiagen GmbH, Hilden, Germany)according to the manufacturer's protocol. RNA was retrotranscribed usingrandom primers and reverse transcriptase (Promega Corp., Madison, Wis.,USA). Quantitative real-time PCR was performed with the aid of ABI 7500Prism system (Applied Biosystems, Foster City, Calif.). The porcinegenes studied were Bradykinin receptor B1 (BDKRB1; Ss03389804_s1,Thermofisher scientific), calcitonin gene-related peptide (CGRP;Ss03386432_uH) and catechol-0-methyltransferase (COMT; Ss04247881_g1) todetect pain marker upregulation, interleukin-6 (IL-6; Ss03384604_u1) toexamine the inflammatory response and brain-derived neurotrophic factor(BDNF; Ss03822335_s1) to determine nerve growth. 18s was used as ahousekeeping gene control.

Histological analysis and immunofiuorescence imaging

Histological analysis was performed on degenerated IVDs harvested at 2,6 and 10 weeks after degeneration. The IVDs were sectioned and stainedusing hematoxylin and eosin for morphological analysis, as previouslydescribed (D. Sheyn, D. Cohn Yakubovich, I. Kallai, S. Su, X. Da, G.Pelled, W. Tawackoli, G. Cook-Weins, E. M. Schwarz, D. Gazit, Z. Gazit,PTH promotes allograft integration in a calvarial bone defect. Mol Pharm10, 4462 4471 (2013). For immunofluorescent staining, tissues weredeparaffinized, and the antigens were retrieved by incubation inpreheated Target Retrieval Solution (Dako, Carpinteria, Calif.) for 45minutes in 37° C. Nonspecific antigens were blocked by applying blockingserum-free solution (Dako). Slides were stained with primary antibodiesagainst BDKRB1, CGRP, COMT, IL-6 and BDNF. The primary antibodies wereapplied to the slides and incubated in 4° C. overnight, washed off usingPBS, and the slides were incubated with secondary antibodies for 1 hourin room temperature, after which they were washed off with PBS (TableS1). Slides were then stained with 4′,6-diamidino-2-phenylindoledihydrochloride (1 μg/ml) for 5 minutes in the dark, after which theywere again washed three times with PBS. A VectaMount mounting medium(Vector Laboratories, Burlingame, Calif.) was applied to the tissue. Theslides were imaged using a four-channel Laser Scanning Microscope 780(Zeiss, Pleasanton, Calif.) with x20 magnification, z-stacking, and 5^(×)5 tile scanning. For zoom-in images, a single z-stacked image wasgenerated. All samples were scanned using the same gain and exposuresettings.

Statistical Analysis

GraphPad Prism 5.0f software (GraphPad Prism, San Diego, Calif.) wasused to analyze the data. Data analysis was conducted using one-way ortwo-way ANOVA with Tukey's multiple comparison post hoc test. Resultsare presented as means±SE. In box-and-whisker diagrams, the median isshown with a horizontal line, the box extends from the 25th to the 75thpercentile, and the whiskers extend from the smallest value up to thelargest. Pearson correlation was performed between qCEST and pH values.ROC curves were generated for qCEST and area under the curve wascalculated. P values that were less than 0.05 were considered to bestatistically significant.

TABLE S1 Antibodies used for immunofluorescence throughout the study.Antigen 1’ antibody 2’ antibody * BDKRBI Anti-BDKRBI antibody DonkeyAnti-Rabbit Alexa TA317572 (OriGene Fluor 488 AffiniPure (cat#Technologies Inc.), 1:100. 711-545-152), 1:1,000. BDNF Anti-BDNFantibody MBS2002795 (MyBioSource), 1:100. COMT Anti-COMT antibody LS-Donkey Anti-Goat IgG B4343 (LifeSpan Biosciences (H + L) Alexa Flour 647Inc.), 1:200. (cat# 705-605-003), 1:1,000. IL-6 Anti-IL6 antibody MAB686Donkey Anti-Mouse IgG (R&D Systems), 1:50. (H + L) ML Rhodamine- CGRPAnti-CGRP antibody ab81887 TRITC (cat# 715-025-150), (Abcam), 1:200.1:1,000. * Purchased from Jackson Immuno Research Laboratories Inc.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosedherein are illustrative of the principles of the embodiments of theapplication. Other modifications that can be employed can be within thescope of the application. Thus, by way of example, but not oflimitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention.

1.-25. (canceled)
 26. A method for treating a subject diagnosed with acondition, comprising: administering a treatment to a subject diagnosedwith a condition, wherein the subject was diagnosed with the conditionby a method comprising: performing a scan of a region of the subject'sbody using a magnetic resonance imaging (MRI) scanner; generating animage of the region of the subject's body from the performed scan usinga quantitative chemical exchange saturation transfer (qCEST) sequence;processing the image to detect one or more physiological biomarkerswithin the image of the region, wherein the physiological biomarkerscomprise a labile proton exchange rate (k_(SW)) between a solute pooland a water pool; and determining that the subject has the condition ifthe labile proton exchange rate is increased relative to a referencevalue, wherein the increased labile proton exchange rate is greater than200 exchanges/second.
 27. The method of claim 26, wherein the increasedlabile proton exchange rate is correlated to a low pH value.
 28. Themethod of claim 26, wherein the increased labile proton exchange rate isfrom 201 to 1000 exchanges/second.
 29. The method of claim 27, whereinthe low pH value is from 5.6 to 6.99.
 30. The method of claim 26,wherein the reference value is a reference labile proton exchange rate,wherein the reference labile proton exchange rate is from 100 to 200exchanges/second.
 31. The method of claim 30, wherein the referencelabile proton exchange rate is correlated to a reference pH value. 32.The method of claim 31, wherein the reference pH value is from 7.0 to7.2.
 33. The method of claim 26, wherein the condition is intervertebraldisc degeneration, discogenic pain, discogenic low back pain, chroniclow back pain, low back pain, back pain, chronic back pain, progressiveintervertebral disc degeneration, osteoarthritis, rheumatoid arthritis,an articular cartilage injury, or temporomandibular disc degeneration,or combinations thereof.
 34. The method of claim 26, wherein the regionof the subject's body comprises a joint or an intervertebral disc. 35.The method of claim 26, wherein the condition is a painful condition.36. The method of claim 26, wherein the increased labile proton exchangerate is correlated with an upregulation of one or more pain-relatedfactors in the subject.
 37. The method of claim 36, wherein the one ormore pain-related factors are bradykinin receptor B1 (BDKRB1),calcitonin gene-related peptide (CGRP), or catechol-0-methyltransferase(COMT).
 38. The method of claim 26, wherein the increased labile protonexchange rate is correlated with an upregulation of one or moreinflammation-related factors in the subject.
 39. The method of claim 38,wherein the inflammation-related factor is interleukin-6 (IL-6).
 40. Themethod of claim 26, wherein the increased labile proton exchange rate iscorrelated with an upregulation of one or more neurogenic factors in thesubject.
 41. The method of claim 40, wherein the neurogenic factor isbrain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF).42. The method of claim 26, wherein the quantitative chemical exchangesaturation transfer (qCEST) sequence is a two dimension (2D)quantitative chemical exchange saturation transfer (qCEST) sequence, ora three dimension (3D) quantitative chemical exchange saturationtransfer (qCEST) sequence.
 43. The method of claim 26, wherein thetreatment is a pharmacological treatment, a biological treatment, a celltreatment, a gene therapy, an interventional surgical treatment, orcombinations thereof.
 44. The method of claim 26, further comprisingdetermining that an origin of the subject's condition is within theregion of the subject's body where the physiological biomarker wasmeasured.
 45. The method of claim 27, wherein the low pH value isindicative of the subject having the condition.
 46. The method of claim26, further comprising selecting the treatment prior to administeringthe treatment.